24th International Ion Chromatography Symposium (IICS 2012)

The 24th International Ion Chromatography Symposium (IICS 2012)

 will take place at Andel's Hotel, Berlin, Germany, on the

The symposim will be chaired by Andreas Seubert from the University of Marburg, Marburg, Germany, and co-chaired by Charles Lucy from the University of Alberta, Edmonton, Canada.  

Ions (and ionic interactions) obviously play an important role in chemistry and ion chromatography is an important technique in the world of separation science. Ion chromatography is capable of analysing large biomolecules as well as small- and medium-sized ions. 

symposium is specifically focuses on the separation and analysis of ions primarily by ion chromatography, but also other techniques, including capillary electrophoresis and other ion-related analytical techniques from electrochemistry and spectroscopy are featured.  

 will provide an overview on actual and upcoming analytical applications from various fields of ion analysis, combined with the latest fundamental developments around new columns, detectors and complete analytical systems. IICS will provide an excellent venue to share your application or your analytical problem related to ion analysis.  

Greg Dicinoski from ACROSS, University of Tasmania, Hobart, Australia, will deliver a short course called an 

Introduction to Modern Ion Chromatography 

which will keep everyone up to speed on the history and advances of this exciting technique on the opening day of the symposium. This will then be followed by the welcome reception. 

Electrodialytic Devices in Chromatography: Past, Present and a Cloudy Crystal Ball 

by Purnendu Dasgupta, University of Texas at Arlington, Arlington, Texas, USA, and two IICS Award Presentions: 

Applying Host-guest Chemistry to Ion Chromatography

 by John Lamb from Brigham Young University, Provo, Utah, USA, and 

Convenient Water Quality Monitoring by Ion-exclusion Chromatography with Multifunctional Separation Mechanism

 by Kazuhiko Tanaka from Hiroshima University, Higashi-hiroshima, Japan. 

include Multidimensional Ion Chromatography; Capillary Ion Chromatography; Smart Materials; Young Scientist Session; Water Analysis Session; Microscale Separations; Food Analysis/Carbohydrate Analysis; Combustion Ion Chromatography, Detection Techniques; Pharmaceutical Analysis; Forensic Analysis; IC–MS; Column Technology and a session on Fundamentals. 

There will be posters and networking sessions throughout the event giving attendees the opportunity to meet with their peers and leading luminaries from the world of ion chromatography.  

To view the final programme agenda and to register, please visit the 

 Californian Separation Science Society (CASSS) 

CoSMoS 2012 - Conference on Small Molecule Science

				Rhode Island Convention Centre, Providence, Rhode Island, USA 			

http://www.cvent.com/events/cosmos-annual-conference/event-summary-7b8db8b280df403085ce7c539aa348ef.aspx

29th International Symposium on Chromatography

Nicolaus Copernicus University Campus, Torun, Poland			

Polish Chemical Society/Commission for Chromatographic Analysis 

SFC 2012 – 6th International Conference on Packed Column SFC

2012 International Light Scattering Colloquium (ISLC)

Four Seasons Biltmore Resort, Santa Barbara, California, USA

http://www.wyatt.com/component/option,com_jumi/Itemid,120/fileid,12/#

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Event News

A liquid chromatography–tandem mass spectrometry (LC–MS-MS) method to determine clenbuterol-like beta-agonist residues in hair was developed and validated. Hair samples were extracted with 0.1 mol/L hydrochloric acid and 0.2 mol/L ammonium acetate buffer (pH 5.0) and cleaned up using a solid-phase extraction cartridge (60 mg/3 mL). Linearity was assessed in the 0.1–50 ng/L (r = 0.9951–0.9996) range for 18 betaagonists in hair samples. The analytical recoveries were acceptable; between 72.76% and 122.22%, with intraday and interday precisions of relative standard deviation (RSD) between 1.6% and 17%, and the limits of detection were all lower than 0.5 ng/L. The proposed LC–MS–MS method has high sensitivity and strong specificity with a relatively easy procedure for sample preparation. Thus, it can be used to detect beta-agonist residues in hair, providing a basis for regulating food safety. 

Beta-adrenoceptor agonists (β-agonists) are used to treat bronchiectasis and to increase pulmonary ventilation. Clenbuterol is the best known of the aniline-like β-agonists. When the drug dosage exceeds the treatment amount by 5–10-fold, it can cause a substantial increase in the muscle mass of animals such as cows, lambs, pigs and poultry. Because the drug has stable chemical characteristics and cannot be destroyed by general heating, it remains in a significant residual amount in edible animal tissue. For example, clenbuterol is stable in 100 °C water and has a half life of 5 min in 260 °C oil (1). When a person eats the animal tissue (for example liver, kidney, lung, or meat), toxicity symptoms may develop (2). Thus, the public representatives of the European Economic Community in July 1997 approved a law to ban the use of β-agonists in all animals destined for human consumption (3). All β-agonists are currently banned from use in edible animals in China, the United States and the European Union. 

There have been few studies on the development of a standard method of assaying nondestructive living samples from animal hair. For this reason, we investigated the development of an assay using hair for the detection of β-agonists.  

Methanol, ethyl acetate, acetonitrile and formic acid were all high performance liquid chromatography (HPLC)–grade (Merck, Darmstadt, Germany). Sodium dihydrogen phosphate, hydrochloric acid, and ammonium hydroxide solution were of analyticalreagent grade. Water was produced by a Milli-Q system (Millipore Corp., Molsheim, France). 

A clenbuterol hydrochloride reference substance was purchased from the National Institute for the Control of Pharmaceutical and Biological Products in China, which was equivalent to 88.38% clenbuterol. Terbutaline sulfate, salbutamol, fenoterol hydrobromide and salmeterol were all in accordance with the 

; ractopamine hydrochloride, brombuterol hydrochloride, cimaterol, clenpenterol hydrochloride, mabuterol hydrochloride, tulobuterol hydrochloride, mapenterol hydrochloride, cimbuterol and bambuterol hydrochloride were all supplied by WITEGA Laboratorien Berlin-Adlershof GmbH (Berlin, Germany); ritodrine hydrochloride, isoxsuprine hydrochloride, and penbutolol hydrochloride were all in accordance with the 

; D9-clenbuterol and labetalol hydrochloride were all supplied by Sigma (Carlsbad, California, USA). 

Hair samples were obtained, both from pigs from the Shanghai hoggery and from people in the outskirts of Shanghai; the hair was processed, washed and measured separately. Hair that tested drug-negative was regarded as a blank hair sample. 

The equipment included an Agilent liquid chromatograph connected to a triple-quadrupole mass spectrometry (MS) detector with electrospray ionization (ESI) sources (Agilent Technologies Corp., Santa Clara, California, USA); another Agilent liquid chromatography system, including a binary pump, degasser, column oven, and autosampler (Agilent Technologies, Waldbronn, Germany); an ultrasonic instrument (Shanghai Kudos Ultrasonic Instrument Corp., Shanghai, China); ultracentrifugation (Thermo Electron Corp., Osterode, Germany); a constant temperature incubator (Shanghai Medical Constant Temperature Equipment Factory, Shanghai, China); a vortex mixer (IKA Corp., Staufen, Germany); a nitrogen blower (Organomation Associates Inc., Berlin, Massachusetts, USA); an electronic balance (Beijng Sartorius Balances Co., Beijing, China); a solid-phase extraction (SPE) manifold (Waters, Milford, Massachusetts, USA); a PCX SPE column (60 mg/3 mL) (Tianjin Agela Technologies Corp., Tianjin, China); an MCX SPE column; and the Milli-Q water purification system (Millipore Corp., Molsheim, France). 

Clenbuterol-Like β-Agonist Standard Solutions: 

Standard stock solutions of clenbuterol and clenbuterol-like β-agonists were prepared in methanol at a concentration of 100 μg/mL and stored at 4 °C. The standard stock solutions were used to prepare 100, 10 and 1 ng/mL standard solutions. 

The internal standard (D9-clenbuterol isotope) was also prepared in methanol at a concentration of 100 μg/mL and stored at 4 °C. The internal standard stock solution was used to prepare a 500 ng/mL internal standard solution. 

 Each hair sample was washed once with 1% polysorbate-80 and three times with ultrapurified water, dried at 40 °C, cut into pieces and stored in a dry place. 

A total of 500 mg of each washed hair sample was weighed, and 100 μL of internal standard solution (500 ng/mL) and 10 mL of 0.1 mol/L hydrochloric acid were added. This mixture was then incubated in a 40 °C water bath for 18 h, removed, cooled to room temperature, vibrated on a shaker for 10 min and centrifuged at 4600 rpm for 5 min. The supernatant was removed and kept, and the residue dissolved in 5 mL of 0.2 M ammonium acetate buffer (pH 5.0). The vortex was mixed for 10 min, and centrifuged. The supernatants were combined. 

A 60 mg/3 mL PCX column was activated sequentially with 3 mL of methanol, 3 mL of water and 3 mL of 2% (w/v) formic acid. The sample supernatant was transferred to the top of the extraction column. The column was washed with 3 mL of 2% (w/v) formic acid solution and 3 mL of formic acid in turn, then dried for 15 min at a pressure of –10 mm Hg. Elution was conducted with 12 mL of 90:3 (v/v) saturated ammonium hydroxide–ethyl acetate. The eluate was collected and dried under nitrogen at 40 °C, reconstituted with 200 μL of 0.1% (w/v) formic acid and then vortexed for 1 min for analysis. 

Liquid Chromatography–Mass Spectrometry Analysis: 

				Chromatography was performed at 30 °C on a 100 mm × 3.0 mm, 1.8-μm 

 HPLC Agilent C18 column at a flow rate of 0.4 mL/min, using 0.1% formic acid aqueous solution as mobile phase A and 0.1% formic acid in acetonitrile as mobile phase B. A total of 10 μL of each sample preparation was injected onto the HPLC column and eluted with a mobile phase gradient: 12%–70% B in 6.5 min and back to 12% B in 0.1 min, with the analytical column equilibrated in 4 min before the next injection. The liquid chromatograph was connected to the ESI MS-MS instrument.  

 The mass spectrometer was operated in the positive ESI mode at a capillary voltage of 4.0 kV, with the ion source set at 350 °C, in a nitrogen gas flow of 11 L/min, nebulizer 35 psi. For each analyte, ion information was acquired under the conditions shown in Table 1. 

Table 1: LCâMS-MS acquisition parameters for the target compounds.			

A 500-mg blank hair sample was  taken. As described above, the chromatography results were recorded and compared with the spiked sample and internal standard. The total ion chromatograms (TICs) showed that the endogenous substances of the hair would not contaminate the test for 18 kinds of β-agonists in the hair (Figure 1). 

Figure 1: (a) Blank hair TIC, (b) TIC of blank hair with internal standard added, and (c) TIC of blank hair with 18  Î²-agonists and internal standard added.			

A 500-mg blank hair sample was weighed and processed to give sample concentrations of 1, 2, 5, 10, 20 and 50 ng/g, following treatment and analysis as described earlier. Internal standard calibration curves demonstrated a linear relationship between the ratio integrated peak area and the internal quantitative ion, at a concentration range of 1.0–50.0 ng/g, correlative coefficient (γ) all above 0.99. 

A 500-mg blank hair sample was weighed following the assay method evaluation and results as set out in the European Community guidelines (4). Hair samples at three standard concentrations (1.0, 1.5 and 2.0 ng/g) of clenbuterol-like β-agonist were prepared and stored at room temperature for 12 h. The samples were treated and analyzed as described in the section on pretreatment of hair samples. Six samples were tested in parallel for three days continuously, and intraday accuracy and interday accuracy were calculated (Table 2).  

A 500-mg blank hair sample was weighed following the assay method evaluation and results set out in the European Community guidelines. Hair samples at three standard concentrations (1.0, 1.5, and 2.0 ng/g) of clenbuterol-like β-agonist were prepared and stored at room temperature for 12 h. The samples were treated and analyzed as described in the section on pretreatment of hair samples. The content and recovery rates were calculated as recovery rate % = determined amount/added amount × 100% (Table 3). 

The limits of detection (LODs), defined as the lowest value that differs from the blank with a 90% of confidence level, were all less than 0.5 ng/g for clenbuterol-like β-agonists. The limits of quantification (LOQs) were all less than 1 ng/g.  

All of the 20 samples of pig hair from the Shanghai hoggery and the 20 samples of human hair were collected randomly from the outskirts of Shanghai, treated as described above and analyzed by LC–MS-MS. The samples of pig hair were not found to contain any clenbuterol-like β-agonists. In the samples of human hair, two kinds of β-agonists were detected: clenbuterol and salbutamol. Six samples contained clenbuterol, with concentrations of 2.87, 1.65, 1.04, 1.30, 14.9 and 4.28 ng/g, respectively; and three samples contained salbutamol, with concentrations of 1.18, 1.69 and 1.55 ng/g, respectively. 

A 1-ng/mL standard solution of clen-buterol-like β-agonist was incubated in 0 °C, 40 °C, 50 °C, 60 °C and 80 °C water baths overnight (18 h), respectively, and allowed to stand to room temperature, then analyzed by LC–MS-MS.The results showed that the content of some the β-agonists (such as salbutamol, fenoterol, isoxsuprine, ractopamine, bambuterol, brombuterol, clenpenterol, salmeterol and penbutolol) decreased at temperatures greater than 50 °C. Therefore, 40 °C was used to hydrolyze the β-agonists present in hair.  

The effects of acid and alkaline hydrolysis on 18 kinds of β-agonists were initially tested. Briefly, 1 ng/mL of β-agonist hydrochloric acid solution (0.1 mol/L) and 1 ng/mL β-agonist sodium hydroxide solution (1 mol/L) were prepared and each placed in a 40 °C water bath overnight for 18 h, removed, cooled to room temperature and subjected to LC–MS-MS analysis. The results showed that concentrations of salbutamol, terbutaline, cimaterol, fenoterol, cimbuterol, ractopamine, isoxsuprine and bambuterol decreased in alkaline solutions. Because the majority of β-agonists were in the form of hydrochloric salts and sulfate salts, acid conditions at 40 °C overnight had no effect. Because of the instability of some β-agonists under the alkaline conditions, acid hydrolysis was chosen for this study.  

: Standard solution was added to five 500-mg portions of blank hair to prepare spiked hair samples of 1 ng/g. Then, 10 mL of 0.1 mol/L hydrochloric acid was added. Each solution was incubated at 40 °C overnight for 18 h, cooled to room temperature, shaken, centrifuged and the hydrochloric acid hydrolysis solution removed. Five successive extractions using 5 mL of ammonium acetate solution (0.2 mol/L, pH 5.0) were performed on the samples. Each of the five extraction solutions was assayed by LC–MS–MS separately, and the chromatographic results are shown in Figure 2. The results showed that the third extraction solution was almost devoid of β-agonists. Thus, after hydrochloric acid hydrolysis of the hair, two extractions with ammonium acetate solution were chosen for this study. 

Figure 2: TICs of five successive extractions.			

				Standard solution was added to four 500-mg blank hair samples to prepare spiked hair samples of 1 ng/g. Then, 10 mL of 0.1 mol/L hydrochloric acid was added. The solution was incubated at 40 °C overnight for 18 h. The samples were cooled to room temperature, shaken, and centrifuged, and the hydrochloric acid extraction solution removed. To the sample, 5 mL of ammonium acetate solution (0.2 M, pH 5.0) was added, and two samples were shaken for 10 min, with another two samples being subjected to ultrasound for 10 min. LC–MS-MS was then carried out separately. The chromatographic results show that the extraction efficiency of shaking was slightly better than that of ultrasound; therefore, shaking was used in the study. 

: SPE is an effective method to purify various β-agonists. It is best applied on aniline and phenol and has various applications to β-agonist residue analysis. In this study, a mixed cation-exchange column was chosen, with double mechanisms of ion exchange and reversed-phase retention. The sample was put into the activated SPE column, and the β-agonist drugs were ionized at pH 5.0 and exchanged with SPE cation chromatography. After impurities were washed out, the β-agonist drugs became deionized and were washed out by ethyl acetate in saturated ammonia water. SPE columns (PCX, Agela, 30 mg, 3 mL) were chosed to purify the 18 kinds of β-agonist in the hair extraction solution for this study. 

				This study evaluated ethyl acetate and ether as eluents. A 1-ng/mL standard solution was added to the activated SPE column. Then ethyl acetate or ether, both in saturated ammonia water, was used to elute the β-agonist drugs retained by the column. The eluate was dried by nitrogen gas, dissolved again in ethyl acetate or ether in saturated ammonia water, and subjected to LC–MS-MS. Ethyl acetate offered better results than ether; thus, ethyl acetate was chosen for this study. 

During sample analysis, retention time and the relative abundance ratio were important basis for qualitative analysis. In this study, two β-agonists, clenbuterol and salbutamol, were found in human hair. Their retention times and relative ion abundances were all within the appropriate range. Therefore, the analytical results were accurate and reliable.  

In this study, a method for detecting 18 β-agonists in hair was established. Till now, there has been no similar report in China and only one report internationally (5). This study simultaneously tested 22 β-agonists in hair using a strong alkaline solution to hydrolyze the hair. After examining the experimental results, it was found that a strong alkaline solution destroys β-agonists (such as salbutamol and others) in the hair, causing the concentrations of many kinds of β-agonists in the hair to decrease, and thus decreasing detection sensitivity. The concentration check of the method is 5 ng/g. To optimize the experimental conditions in this study, a mild acid solution was chosen to hydrolyze hair, and referring to the EU regulations, the lower concentration (1.0, 1.5, and 2.0 ng/g) hair samples were examined by the methodology. The detection limit was lower than 0.5 ng/g, and the established method had high sensitivity, strong specificity, rapid response, high accuracy and was suitable for testing various kinds of β-agonists in hair. 

 is with the department of pharmacy, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. 

 are with the Shanghai Institute for Food and Drug Control, Shanghai, China. 

Drug Residues in Foods: Pharmacology, Food Safety, and Analysis

Analysis of the Residues of the Animal Drug [M], (

Shanghai Science & Technology Press, Shanghai, China, 2002), pp. 641–682.  

(3) 9-EEC Directory 96/23/CE, Off. J. European Community, I.125 (1996) 10.  

(4) EC Commission Decision 2002/657/EC, Off. J. European Communities L22⅛ 14 August, (2002).  

Article

A sensitive and selective liquid chromatography spectrometry mass spectrometry (LC–MS–MS) method to determine clenbuterol-like beta agonist residues in human hair was developed and validated.

Determination of Clenbuterol-Like Beta-Agonist Residues in Hair

This instalment provides a brief overview of some multidimensional approaches targeted at intact protein analysis for life science applications, particularly for biopharmaceutical analysis. 

Since the 1944 publication of a paper by Consden, Gordona and Martin (1) describing multidimensional separations, the prospect of using this technique to address complex analytical challenges has sustained the interest of the analytical chemistry community. Analysts often use multidimensional techniques when they require higher separation capacity than single-dimension analyses can afford. By using dimensions of various selectivity for complex analytes such as food, polymers and hydrocarbons, maximum resolution can be achieved among components that are generally difficult to separate using a single-dimension technique. 

The multidimensional approach for separations has been — and continues to be — highly utilized. Not surprisingly, its use as an analytical technique has been expanded. It now includes various applications that do not necessarily result in greater resolution. In those applications, the technique serves as the means for conducting separate analyses of the same analyte without the need for multiple instruments. It can also reduce steps for sample preparation and provide a convenient way to perform automated assays. 

In the past few years, an emerging area of focus in the pharmaceutical industry is the biopharmaceuticals. The compounds in this class are very complex and require a high degree of characterization. Indeed, the level of characterization necessary is increasing, largely because of improved analytical technologies and regulatory requirements. What's also at play is the emergence of biosimilar approval pathways, in which protecting intellectual property for innovators or proving similarity for biosimilar producers is increasingly important (2).  

Developing and manufacturing biopharmaceuticals requires amassing a variety of chromatographic and mass spectral data. The different chromatographic modes probe particular characteristics of the analyte. Some of these characteristics include size, charge and mass profile. As such, they are related to, or are themselves, critical quality attributes. 

For some biopharmaceutical applications, a multidimensional separation can reduce a practitioner's analytical burden, yielding greater assay reproducibility by dispensing with hands-on sample preparation. Note, however, that although adding chromatographic dimensions can prove useful, to do so without realizing additional benefit, such as increased resolution, sensitivity,or automation, introduces unnecessary complexity.  

This column instalment provides a brief overview of some multidimensional approaches targeted at intact protein analysis for life science applications, particularly for biopharmaceutical analysis. For the applications described here, adding a liquid chromatography (LC) dimension automates sample preparation and provides a convenient means of analysing an analyte by different methods that require different chromatographic eluents. As such, these applications are meant for targeted analyses where sample complexity does not demand adding a dimension. Rather, the criterion for needing a multidimensional separation is the primary chromatographic method's incompatibility with the desired detection method. For example, because of high mobile-phase salt concentrations, ion-exchange chromatography (IEC) is incompatible with mass spectrometry (MS) detection directly. Capturing a peak of interest to a second-dimension reversed-phase column, however, allows for desalting the analyte and collecting MS data.  

A convenient way to perform a multiple-dimension analysis is to collect fractions from one dimension, and then reinject those fractions onto the next dimension. Often, the first-dimension separation serves as a sample-preparation step, for example, an affinity purification using protein A. Yet fraction collection of a sample separated by IEC or sizeexclusion chromatography (SEC) is also commonplace (3). Moreover, fraction collection provides a convenient means to comprehensive analysis, regardless of the chromatographic run time for each dimension because the fractions can be stored in a temperaturecontrolled environment. 

One type of first-dimension separation based on affinity purification serves to enrich the collected fraction in the analyte of interest in an automated fashion. The method used is often similar, if not identical, to the method that will be used when purifying the molecule during the production process. During an affinity purification using protein-A, a UV detector monitors the column eluent, and the resultant peak area can be used to assess a protein titer. Following UV detection, the purified material is collected using a fraction collector. The fraction collector is usually temperature controlled to prevent sample degradation. A significant benefit of an affinity purification step lies in its highly reproducible peak elution, allowing the generation of a generic method suitable for a variety of analytes (4). 

After the first-dimension purification, the collected fractions (or fraction) are reinjected for subsequent analyses, such as IEC (to access charge heterogeneity) or SEC (often to access aggregation and uniformity of the molecular size). With limited handling, a single sample can be purified and analysed using various second-dimension methods, without the need for multiple chromatographic systems. Commercial instruments are available for these types of analyses in an automated fashion; they enable users to define the purification conditions and methods for each of the secondary analyses for each sample.  

Another type of first-dimension separation for intact proteins relies on the first dimensions to be more highly resolving, such as IEC or SEC. Fractions can be collected in two ways: by a timed event, with equal fractions collected across either part or all of the chromatographic separation, or by detection thresholds for collection, which limits the number of fractions collected. In either collection regime, each collected fraction undergoes a single second-dimension analysis or, potentially, multiple second-dimension analyses. 

This type of protocol is useful to identify and characterize low-level impurities present in a sample. For example, an SEC analysis for a sample clearly indicates that the sample contains aggregated species, evidenced by a peak correlating to a molecule roughly twice the size of the desired product. One can easily adopt a multidimensional approach by collecting the suspected aggregated species. The collected fraction could then be analysed by an orthogonal LC method to obtain the distribution of components in the aggregated species. The second-dimension separation can be done with IEC or a reversed-phase-MS analysis. The IEC analysis can help determine whether the species is enriched or deficient in charged species. The reversed-phaseMS analysis can help determine the presence of species with different masses in their monomeric forms. 

A potential problem associated with the fraction-collection technique arises when it is used for lowabundance species, often the target of this type of analysis. It is common for low concentrations of protein species to adsorb to the walls of collection vessels, which can impede the ability to conduct a second-dimension analysis. Nevertheless, one can avoid this phenomenon by prudently choosing a collection vessel, assuming a suitable vessel exists for the particular application (5). Alternatively, you can conduct multiple first-dimension separations and collections to ensure the retention of sufficient analyte for subsequent analysis. 

An alternative multidimensional approach adopts an on-line collection scheme where fractions, or "heart cuts," collected from the first dimension are captured directly onto a second chromatographic column for the second-dimension analysis. Life science applications have used multidimensional approaches for some time; for example, Washburn and colleagues (6) conducted proteomics studies using multidimensional analysis. Recently, multidimensional separations have also helped to identify host-cell proteins during biopharmaceutical development (7). 

As with the off-line fraction-collection techniques, on-line multidimensional separation proves useful when investigating impurities that are insufficiently resolved from other components in a complex mixture in a single-dimension LC analysis. In addition, on-line approaches are useful for investigating low-level impurities, because analytes are less susceptible to loss upon collection. In either case, a multidimensional approach allows analysts to either reduce sample complexity or enhance an assay's dynamic range. One particular advantage of the on-line approach is that analytes can be stored at physiologic conditions until just before an analysis.  

A common application of an on-line, multidimensional approach that is similar to off-line fraction-collection techniques involves purifying an antibody using protein A followed by an orthogonal approach. Emphasized here is the coupling of a first-dimension, protein-A purification followed by reversed-phase-MS analysis of the heart cut. Each of these analyses are commonly performed, often separately, during the development and production of antibodies, but with modern instrumentation they can be accomplished sequentially on a single LC–MS system. Other second-dimension analyses exist, and their utility in a heart-cut scheme equals that of the off-line fractioncollection methods.  

In the application example discussed below, the second-dimension separation is a low-resolution separation conducted on a short column — one generally used for trapping and eluting analytes. Although higher-resolution separations can be performed in the first dimension, second dimension or both, one must consider overall system-pressure restrictions when both columns are placed in-line. 

The production and formulation of antibodies intrinsically results in mixtures that contain excipients that are not compatible with MS analysis. Particularly in the case of MS analysis, efficient removal of these components is imperative for achieving sufficient MS sensitivity. Hence the attractiveness of affinity purification, which efficiently removes excipients by selectively binding only the analyte of interest while the remaining components are eluted.  

The remainder of this article discusses in detail a specific application example that focuses on the challenge of coupling purification with mass profiling of antibodies using an on-line, multidimensional LC system. The methods were developed on an LC system with 2D technology coupled to a quadrupole time-of-flight (Q-TOF) mass spectrometer.  

Figure 1 depicts a plumbing diagram with an affinity purification column configured for conducting the heart cut. The configuration provides two valve positions. In position 1, the loading position, the first-dimension column is loaded. Eluent flows through a UV detector and then to a waste vessel. Diverting the eluent to waste is necessary to minimize unwanted analytes (such as components from cell culture media) from collecting on the reversed-phase column. During the loading step, the 2D LC pump delivers initial gradient conditions to the second-dimension column. After removing unbound components, the antibody elutes from the affinity column. During the first-dimension elution phase, in an actuation sequence known as "heart cutting," the valve switches to position 2, diverting a portion of the first-dimension eluent to the second-dimension column. 

Figure 1: Example plumbing diagram for on-line heart-cutting analyis. 

After collection from the first dimension, the valve returns to position 1, and the second-dimension gradient is initiated. Because each dimension is used in series for analysis, the overall run times for each dimension (either protein A or mass measurement by reversed-phase-MS analysis) is reduced compared to running either dimension individually off-line. The run time is decreased because equilibration time for each dimension can mainly be accomplished while the other dimension is conducting an analysis.  

Figure 2(a) shows that the on-line affinity purification is highly reproducible, as evidenced by an overlay of 12 replicate injections with a relative standard deviation (RSD) of 0.078%. In addition, these methods provide a linear response based on mass load, which is shown in Figure 2(b). One would expect similar results using an off-line fractioncollection system. 

Figure 2: Representative UV chromatograms for purification of mAb by protein A. 

An important consideration, besides chromatographic reproducibility, is heart cutting through an on-line method. Making appropriate choices in valves, valve event timing and ensuring optimal plumbing connections are all important factors to consider when designing the experiment. Because collection is generally based on a timed event or by a specified threshold, such as UV intensity, the valve trigger events must be reproducible. One must also account for any delay time in the system between the point at which a desired peak is detected and its subsequent collection. Particularly, note that the flow rate, tubing length and the internal diameter all affect valve timing.  

A first-dimension protein A purification for a monoclonal antibody (mAb) is shown in Figure 2(a). The highlighted area represents the time selected for peak collection. The area was defined by timed events to actuate the left-hand valve (shown in Figure 1) to move to position 2 at the start of collection and then back to position 1 at the end of collection. The collected peak is focused on the second-dimension column and quickly desalted before elution. A valve on the mass spectrometer diverts the remaining eluent stream to waste. After sufficient removal of salt and buffer, primary components of the protein-A effluent, the desalting valve diverts the flow to the mass spectrometer's source, a steep gradient runs and the analyte is eluted as a concentrated peak. 

Figure 3 shows that the second-dimension separation and peak collection are reproducible for a variety of mass loads, as evidenced by the total ion chromatograms for the MS analysis.  

Figure 3: Overlay of total ion chromatograms for an antibody loaded on a first-dimension protein A column, collected and subjected to MS analysis. Each analysis was run in triplicate. 

An on-line approach can also provide mass-spectral information for analytes separated by IEC and SEC. As mentioned previously, one could also use off-line fraction collection to perform this type of analysis. Note, however, that some low-abundance species can adsorb to collection-vessel walls, impeding or inhibiting your ability to derive meaningful data from the collected fraction.  

Adopting an on-line approach is one way to overcome the challenge posed by adsorption. One plumbing scheme for an on-line analysis, identical to that shown in Figure 1, allows the analysis of one fraction per first-dimension analysis. To analyse additional fractions, one must configure a system that can collect fractions onto separate trapping columns or a system that can hold fractions in loops before analysis. Though the latter configuration confers the advantage of requiring fewer overall experiments, system configurations can rapidly become very complex. 

This column instalment describes various methods for using a multidimensional approach, including on-line and off-line methods. Such methods can simplify sample analysis in either of two ways: by reducing hands-on sample preparation or by replacing significant analytical challenges with routine ones. Moreover, spurred by practitioners' increased interest in multidimensional techniques, manufacturers of analytical instruments are providing greater flexibility to configure multidimensional systems. As we increasingly adopt these technologies, systems particularly suited for performing multidimensional separations will likely emerge to make analysts lives considerably easier. 

is a principal scientist at Waters Corporation (Milford, Massachusetts, USA). He received his PhD in inorganic chemistry from the University of Vermont, in Vermont, USA, in 2005. Following this he was a postdoctoral fellow in the pathology department at the university. Most recently his work has focused on biopharmaceutical characterization using LC and LC–MS technologies. 

 joined Waters in Milford, Massachusetts, USA, in 1998. She has a wealth of experience in applying LC–MS technologies into various application fields such as metabolite identification, metabolomics, quantitative bioanalysis, natural products and environmental applications. Direct correspondence about this column should go to "MS: The Practical Art", 

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

(1) R. Consden, A.H. Gordan and A.J.P. Martin, 

http://www.fda.gov/Drugs/DevelopmentApprovalProcess/HowDrugsareDevelopedandApproved/ApprovalApplications/TherapeuticBiologicApplications/Biosimilars/default.htm

(3) V. Faca, S. Pitteri, L. Newcomb, V. Glukhova, D. Phanstiel, A. Krasnoselsky, Q. Zhang, J. Struthers, H. Wang, J. Eng, M. Fitzgibbon, M. McIntosh and S. Hanash, 

(5) J.M. Mathes, "Protein Adsorption to Vial Surfaces — Quantification, Structural and Mechanistic Studies", Ludwig Maximilian University of Munich (2010). 

(6) M.P. Washburn, D. Wolters and J.R. Yates, 

(7) C. Doneanu, A. Xenopoulos, K. Fadgen, J. Murphy, S.J. Skilton, H. Prentice, M. Stapels and W. Chen, 

Column: MS- The Practical Art

This instalment provides a brief overview of some multidimensional approaches targeted at intact protein analysis for life science applications, particularly for biopharmaceutical analysis.

Multidimensional LC Approaches for Intact Protein Biopharmaceutical Characterization

HPLC 2012 was held 16–21 June in Anaheim, California, USA, for the first time in its long history. In this instalment, coverage of some of the technology and application advances is reported. We review the overall liquid-phase chromatographic trends observed at the symposium, the opening plenary session, awards presented, column technology highlights and sample preparation and detector usage.

The 38th International Symposium on High Performance Liquid Phase Separations and Related Techniques, which alternates between Europe and North America with occasional side meetings in Australasia, was held 16–21 June 2012, in Anaheim, California, USA, for the first time (not far from Disneyland). More affectionately known as HPLC 2012, the symposium is the premier scientific event for bringing together the myriad techniques related to separations in liquid and supercritical fluid media. Chaired by Professor Frank Svec of the Lawrence Berkeley National Laboratory in Berkeley, California, USA, HPLC 2012 assembled 830 participants from all over the world. This number included vendor representatives (150) from 52 exhibitors for the three-day instrument, software and consumables exhibition. Based on the lower number of attendees and exhibitors compared to HPLC 2011 in Budapest, the worldwide economic crisis may have played a role in the support for this important conference.  

The five-day-plus event had a total of 117 oral presentations in plenary and parallel sessions and 443 posters in sessions with 19 different themes. Attendees had their hands full deciding how to allocate their time with an ample social event schedule including a symposium banquet at Disney California Adventure, nine vendor workshops, six "sunrise" tutorial educational sessions and seven short courses (held during the previous weekend). The tutorials were particularly well attended and covered current topics such as organic monoliths, selectivity optimization, hydrophilic interaction liquid chromatography (HILIC), higher order protein structure, capillary LC and ion chromatography (IC) and supercritical fluid chromatography (SFC). 

Trends in Liquid Phase Technology and Techniques

Obviously, high performance liquid chromatography (HPLC) was the predominant technology in the technical sessions at the symposium, but sample preparation, the use of electrophoretic techniques (mostly in a capillary format) and an increase in method development and method transfer papers were strongly evident. From a perusal of the poster and oral presentation abstracts and notes from my colleagues, I broke down some of the major areas of coverage in this year's symposium. These tables are useful to spot trends in the technology, applications of liquid phase separations and detection principles that were introduced in this series. 

Table 1 provides a rough breakdown of the coverage of liquid-phase technology and techniques in the separation sciences. Compared to HPLC 2011 (1), some slight shifts in technology emphasis were noted. Again this year, new developments in column technology led the pack by far with more than a third of the oral and poster papers dealing with new phases and formats. Surprisingly, nearly 40% of the columns' papers dealt with monoliths. Although not yet considered a commercial success, research interest in this technology is still running high, especially in academia. The polymeric monolith segment is less covered by intellectual property rights than the silica monolith segment. Second-generation silica gel–based monolith commercial products feature better efficiency but slightly higher pressure drops because of the change in the macropore/mesopore domain ratio. Presentations on polymer-based monoliths outnumbered silica-based monoliths by a 2.5:1 margin. This surprisingly high margin may have reflected the bias of the symposium chair, who is a leader in this area of polymeric monolith technology. However, a continuation of new developments in polymeric monoliths devoted to the separation of small molecules has shown improvements in column efficiency; originally, the small molecule domain was for the silica-based monoliths and polymeric monoliths were thought to be useful for macromolecules only. 

Table 1: HPLC 2012 papers presented by technology or technique. 

Three other "hot" areas in column technology this year were  

More emphasis on the new breed of superficially porous packings (SPP, also referred to as shell particles, poroshell and fused-core packings) that rival the sub-2-μm particles in terms of column efficiency, but with substantially lower pressure drops. The poster and oral papers referring to SPP outnumbered those devoted to sub2μm totally porous particles by 60%. 

The widespread attention to HILIC for the separation of polar analytes that are weakly retained by reversed-phase chromatography. This year, just for fun, I tabulated the various LC modes reported on at HPLC 2012 (Table 2). Clearly, as expected, reversed-phase chromatography was the number one application mode but HILIC was a fairly strong number two. 

The incorporation of nanoparticles (embedded) into packing media seemed to generate a lot of research interest, especially in polymeric media where the use of gold and carbon nanoparticles imparted higher surface areas (therefore increased capacity) and enhanced selectivity. The nanoparticles themselves can be modified 

 with various functional groups and then used for separations of proteins and other molecules by selective interactions. 

Table 2: HPLC modes represented at HPLC 2012. 

The interest in new research into sub2μm porous packings has waned a bit this year, probably because these columns are now firmly established in the ultrahigh-pressure liquid chromatography (UHPLC) world with more than 30 vendors supplying these products. On the other hand, there were considerable discussions about the practicality of whether the next generation of smaller totally porous particles or superficially porous particles will approach the 1-μm dimensions. 

Sample preparation technology was well represented in the poster papers and two oral sessions were devoted to some of the current techniques. Most prominent were improved solid-phaseextraction (SPE) and solid-phase microextraction (SPME) technologies with new phase chemistries: monoliths, immobilized enzyme 

 reactors, molecularly imprinted polymers (MIPs), dispersiveSPE phases for QuEChERS (quick, easy, cheap, effective, rugged and safe) and mixed-mode phases providing new selectivities.  

Electro-driven separation techniques (for example, capillary electrophoresis (CE), capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC) and isoelectric focusing (IEF) showed a slight drop-off compared to HPLC 2011 (1), but continued to see a strong following in the application papers with great strides being made in interfacing to mass spectrometry (MS). The developing interest in biopharmaceuticals has spurred a renewed interest in CE technologies for the separation of biomolecules. A continued lack of interest in capillary electrochromatography (CEC) was noted with only nine presentations at HPLC 2012.  

Table 3 is a breakdown of the most popular application areas reported at HPLC 2012. Oral and poster presentations on proteomics, biomarkers, protein and peptide separations and identification again constituted about one-quarter of all the papers presents. Within these life science domains, areas of major applications were the separation and study of glycosylated proteins (glycans), monoclonal antibodies and antibody fragments, protein aggregates (size-exclusion focus here) and the continuing search for biomarkers with LC–MS–MS leading the way. The glycans are cleaved from the glycoproteins by specific enzymes. Glycan analysis using carbon-based packing materials saw a number of presentations. Pharmaceutical and biopharmaceutical assays in drug discovery, therapeutics, formulations and active pharmaceutical ingredients (APIs) had wide coverage this year, as usual in the number two position. Other life science applications include the areas of nucleic acids, DNA and oligonucleotides as well as oligosaccharides and amino acid analysis. Another application area with a strong showing was the use of liquid-phase techniques in foods, beverages and food safety.  

Table 3: Papers presented by application area. 

In this instalment, I present some additional scientific highlights from HPLC 2012, including the various awards and honorary sessions that took place. Because it was virtually impossible for one person to adequately cover all oral and poster papers, my coverage will somewhat reflect a personal bias; I was able to get presentation notes from some of my colleagues, who are acknowledged at the end of this column instalment. 

 For the seventh year in a row, the Horváth award sessions, named for the late Professor Csaba Horváth, one of the founders of this series and a mentor of young scientists, were featured. This award, supported by HPLC, Inc., a nonprofit group under the guidance of the Permanent Scientific Committee, was established for young scientists in the separation sciences under the age of 35. The award is based on the best oral lecture presented in the Horváth sessions, and the winner is selected by a jury named by the Permanent Scientific Committee. The award consists of a cash prize, invitation to present an oral at HPLC 2013 and a crystal trophy. This year there were 10 nominees, all with strong research credentials. This year's winner was Stefan Bruns of the Philipps-Universität in Marburg, Germany (Figure 1). His coauthor and professor of this awardwinning presentation was Ulrich Tallarek. The title of the paper was "Slurry Packing Parameters and their Influence of Capillary Column Morphology." Slurry packing of particles in LC columns has always been considered to be a form of "black magic," and this research group set out to provide more of a scientific basis for determining the packing microstructure of columns with a focus on the newer superficially porous (core–shell) particles. Using simulations of dispersion to establish a morphology–transport relationship, the workers used confocal laser scanning microscopy to reconstruct segments of capillary columns packed with SPP materials. Using slurry packing, they experimentally investigated particle size distribution, particle surface roughness, column inner diameter and how these variables affected packing morphology and separation efficiency. The results showed that transcolumn eddy dispersion was of paramount importance and furthermore showed strong evidence of an improved packing structure at the particlecolumn wall interface which reduced the impact of wall effects. 

Figure 1: The 2012 HorvÃ¡th award winner, Stefan Bruns (left) of the Philipps-UniversitÃ¤t in Marburg, Germany, with John Frenz (right) representing the Permanent Scientific Committee. 

 The mainstay of HPLC 2012 was the poster sessions where more detailed applications and methodology studies were reported, often in very specific areas, and face-to-face discussions with the authors were conducted. Fortunately, many of the poster authors were kind enough to provide small reproductions of their poster papers that could be taken for later perusal. Some authors collected business cards and addresses for sending poster reprints by mail or e-mail. Compared to HPLC 2011 (1), the number of posters was greatly reduced, roughly 40% fewer. Reports were that the numbering system was a bit confusing so viewers sometimes had to hunt for the posters of interest. Despite this slight problem, the posters were up for three days, which gave viewers plenty of time to find them. It was noted that about 10–15% of the posters indicated in the abstract booklets were "no shows." 

The poster committee cochairs at HPLC 2012 were Professor Peter Schoenmakers, a member of the Permanent Scientific Committee from the University of Amsterdam in The Netherlands, and Dr Gerard Rozing of Agilent Technologies in Waldbronn, Germany. The 59 members of the poster committee devoted a great deal of time and worked very hard to narrow down the huge collection of posters by the end of the third day to 30 finalists. Eight new reviewers were chosen to help select the 10 winners by Thursday afternoon of the symposium. The selection criteria were based on three factors: inspiration (creativity, newness, uniqueness and originality), transpiration (experimental execution and completeness of work), and presentation (overall readability, visual impression and the author's explanation). The winning posters were viewed by all of the final eight committee jurors. 

Figure 2: Best Poster Award winners (left to right): Elisabeth Haller, University of Vienna (Vienna, Austria); Benjamin Rogers, Purdue University (W. Lafayette, Indiana, USA); Franta Foret (for Karel Kleparnik), Institute of Analytical Chemistry ASCR (Brno, Czech Republic); Gerard Rozing, Agilent Technologies (Waldbronn, Germany) and co-chairman of the Poster Committee; Siyang Li, The Barnett Institute, Northeastern University (Boston, Massachusetts, USA); Jiri Urban, University of Pardubice (Pardubice, Czech Republic); Axel Vaast, Vrije Universitiet Brussel (Brussel, Belgium); Michelle Camenzuli, University of Western Sydney (Parramatta, Australia); Peter Schoenmakers, University of Amsterdam (Amsterdam, The Netherlands) and co-chairman of the Poster Committee; Jana Krenkova, Institute of Analytical Chemistry ASCR (Brno, Czech Republic); Arianne Soliven, University of Minnesota (Minneapolis, Minnesota, USA); and Siarhei Khirevich, Philipps-Universitat (Marburg, Germany). 

The Best Poster Awards, sponsored by Agilent Technologies, were announced at the closing session. The Poster Award winners for HPLC 2012 are shown in Figure 2 and their poster titles are listed in Table 4. All of the winners received a $500 cash prize. Because of the large numbers of awards granted, a detailed technical coverage of each of the award-winning posters cannot be provided. It suffices to say that all the winners should be proud of their accomplishments because they represent the top 2% of all posters presented at HPLC 2012. 

Table 4: Best poster winners at HPLC 2012. 

Opening ceremony plenary lectures, presented on the first evening of the symposium, are supposed to be cutting-edge, thoughtprovoking presentations that inspire attendees to think outside the box. After the "Welcoming Remarks" by Dr Frank Svec, two notables in the field of biopharmaceuticals presented plenary lectures. The first speaker was Steven Kozlowski of the United States Food and Drug Administration's (FDA) Centre for Drug Evaluation and Research (CDER) in Silver Spring, Maryland, USA. His talk was entitled "Regulatory Challenges for Biologics: Biosimilars, Characterization & QbD" and covered the regulatory aspects of biosimilars. CDER's best-known job is to evaluate new drugs before they can be sold, including biosimilars. The second talk was by Professor Barry Karger of the Barnett Institute at Northeastern University in Boston, Massachusetts, USA. Karger spoke about recent advances in LC–MS for translational regulatory science. 

Both plenary talks highlighted the need for effective and predictable regulatory characterization pathways. With more than 36 preinvestigational new drug (IND) requests for proposed biosimilars alone, both speakers discussed the many challenges associated with careful classification of therapeutics and the need for developing robust methods and protocols for compliance. Karger's talk was more integrated with LC–MS and regulatory requirements, and Kozlowski focused on approaches considered by the FDA in ensuring pharmaceutical quality such as quality by design (QbD), current good manufacturing practices (cGMPs) for the 21st century and the process analytical technology initiative. He further defined a desirable biosimilar as having no clinically significant difference between it and the biological drug although a slight difference in rate of occurrence in adverse effects will not make it a dissimilar drug. Subtopics in their talks such as "How close is close enough?" and "Structural and functional characterization pyramids" are a sampling of how challenging this analytical science is compared to traditional small-molecule discovery. One example indicated more than 30000 potential structure modifications on an antibody alone. It is clear that biotherapeutics characterization will require myriad analytical techniques depending on the discovery and manufacturing path and that the interest and effort towards biopharma characterizations will continue to grow and expand.  

The remainder of bio-oriented oral presentations and posters at HPLC 2012 supported the opening plenary talks by displaying very comprehensive characterization methods among many modes, but primarily sizeexclusion chromatography (SEC), reversed-phase chromatography and ion-exchange chromatography (IEC). The extensive coverage on MS detection and characterization of biomolecules was evident in numerous presentations. 

As seen in Table 1, the development and study of columns and stationary phases still dominates new technologies. Both oral sessions and poster sessions primarily devoted to this area included sessions on packed column technologies (3 sessions), monoliths (3 sessions), particles and highly ordered structures, ion chromatography, modeling and theory, HILIC, imprinted sorbents, ionic liquids and chiral separations. If one combines all papers pertaining to column technology, more than a third of the presentations at HPLC 2012 covered this topic. Despite all the advances made in column technology to date, investigations on further developments in academia as well as the commercial side are still taking place. 

From observations made at HPLC 2012, a continuing "hot topic" in HPLC column technology is the discussion of the alternative approaches for developing faster separations and generating more column efficiency at lower pressure drops and which approach will be favoured in the long run. Two years ago, the focus was on comparing sub-2-μm totally porous columns to porous particles in the 2–3 μm range and larger. One year ago, the discussions were focused on the superiority of superficially porous columns over sub-2-μm particle columns. This year's focus was mostly on the superficially porous particles (SPP) themselves since they provide the efficiency of sub-2–μm particles but at around half the operating pressure. I counted a total of 47 papers devoted to SPP columns. There is still some confusion about the nomenclature, and during HPLC 2012, these particles were referred to as SPP, fused-core, shell, porous shell, fused-core and poroshellparticles. It is now generally accepted that SPPs bring the greatest advantage to users compared to porous particles. Monolithic columns, which if improved further, could become a major competitor to the SPP columns. The only question now is: Should both larger and smaller (perhaps down to 1-μm 

) SPPs in various pore sizes and different selectivities be made available? For example, it was demonstrated in the paper of Way and Santasania (2) that 5-μm SPPs provided 60% more efficiency than totally porous 5-μm particles. Table 5 gives an update on the current status of commercial SPP products as of HPLC 2012 for large and small molecules. 

Table 5: Commercial superficially porous particles - status as of HPLC 2012. 

Although users who have already purchased UHPLC equipment are less concerned about the lower pressure drops of these columns, for difficult separations long columns and lots of theoretical plates are always in demand. Therefore, pressure availability is always a concern. Currently, the SPP columns on the market do not have the upper pressure limits of some of the UHPLC instruments but that should change in the near future. 

Several interesting papers on the SPP approach were presented during the packed column technologies sessions. A Monday morning presentation by Georges Guiochon and Fabrice Gritti of the University of Tennessee in Knoxville, Tennessee, USA, discussed the challenges encountered in performing ever faster, more efficient, yet practical separations. Professor Guiochon, who has probably studied the performance characteristics of SPP more than most researchers, focused on describing the ultimate limits of performance that could be expected for smaller particles with a particular focus on SPP. Using small molecules and small proteins (up to 20 kDa) and macromolecules (200 kDa and higher) as model compounds, he described the linear velocities and plate heights that could be expected if particle diameters were to go down as low as 1 μm with "crust" layers with pore sizes in the 150 Å and 300 Å ranges. The latter size provides a sufficient pore size for macromolecule separations. He concluded that the necessary linear velocities for small molecules would generate pressures at 4000 bar or higher and that it would not be practical at the typical linear velocity of 2 cm/s, but for intermediate (0.1 cm/s) or low velocities (0.01 cm/s), the latter preferable for macromolecules, the smaller particles would be practical and possible. In addition, the heat friction generated at higher flow rates and pressures may dominate the separation mechanisms, giving rise to reproducibility problems. The problems of efficiently packing particles in the 1-μm range may also cause headaches. Guiochon called on industry to address the instrumental extracolumn dispersion that may limit the performance of the nextgeneration columns. Short, simple channels to connect the injector–column–detector combination is paramount to good efficiency. He further called on industry to increase data acquisition rates in advance of the coming smaller particle sizes. 

A follow-up talk by Monica Dittmann and co-worker Konstantin Choikhet from Agilent Technologies in Waldbronn, Germany, further addressed the problem of extracolumn contributions to small and microbore HPLC columns in both isocratic and gradient separations. To experimentally determine the dispersion in individual system components such as connection capillaries, detector cells or injection devices, the authors designed an ultralow-dispersion system with a system variance of 0.2 μL. This system also allowed the determination of column-generated peak variances, keeping external contributions at an ultralow level. As might be expected, the extracolumn band broadening was more significant for low 

 values of 10 and higher no significant differences in peak efficiency among various column diameters were noted. The dispersion contributions of various connection capillaries, detection cells and autosamplers were determined as a function of flow for different solvent compositions. Showing that some of the measurements change with flow rate, she concluded that systems must be carefully plumbed with the smallest practical internal diameter tubing (with smaller internal diameters resulting in considerable back pressure despite improved dispersion) and flow cells, and also stating that for isocratic, 1-mm i.d. separations the situation was inevitably the worst case, but 2.1-mm i.d. gradient work was far less challenging.   

The possibility of even smaller SPP particles was explored by Tivadar Farkas of Phenomenex in Torrance, California, USA. Although the feasibility of constructing SPP particles with diameters of 1–1.2 μm has been shown, formidable challenges remain. Firstly, the column pressure would be approximately 2.3 times higher than with the smallest currently available 1.7-μm 

 SPP columns, which operate with the same pressure drop as 1.7μm totally porous particles (TPP). Reproducibly packing efficient columns may be a challenge. Frictional heating would increase by roughly the same 2.3 times, although the heat capacity of SPP has been shown to increase compared to that of TPP of the same diameter. Most likely, elevated temperatures would be required to decrease mobile phase viscosity and the operating pressure. Extracolumn effects of current instruments would need a considerable reduction to realize the true efficiency of the powerful columns. Nevertheless, Farkas showed some van Deemter curves of columns with convincing data that reduced plate heights in the 1–2 range might be achieved, giving something like 500000 plates/m but with an astronomical pressure drop. 

A separate paper by Fabrice Gritti discussed the impact of eddy dispersion on column performance. For modern packed-bed columns like SPP and sub-2-μm columns, eddy dispersion accounts for as much as 75% of the total plate height with mass transfer and axial diffusion contributing much less than originally thought. Eddy dispersion accounts for the contribution to band broadening originating from all sources of flow unevenness in the mobile phase percolating through a column. Gritti's practical and theoretical studies showed that eddy dispersion is mostly controlled by end and wall effects. Using SPP spherical packings with narrow particlesize distributions that are well packed in smooth-walled columns might help to lower eddy dispersion effects and, when used with the future wave of low-dispersion instruments, could result in better performance for everyone. 

An interesting paper by David McCalley from the University of West of England in Bristol, UK, and colleagues compared overloading behaviour of strongly acidic and basic probes for both SPPs and sub-2-μm TPPs in reversed-phase LC. Despite the shell particles containing a smaller volume of porous packing, similar overloading behaviour was found for both materials. The authors studied low to high solute concentrations at different pH. All the columns gave good peak shapes with small concentrations, but ionized acids and bases gave rapid overloading of all columns that was dependent on pH. Some interesting and perhaps unexplained observations were noted such as 4.6-mm i.d. columns giving lower loading than 2.1-mm i.d. columns and an increase then a decrease in efficiency with sample loading for basic compounds at mid-pH. The latter explanation may be related to the presence of ionized silanols at mid-pH values. 

The very first modern widepore SPP was the Poroshell 300 stationary phase (Agilent Technologies) introduced by Jack Kirkland in the early 2000s. These SPPs proved useful for the fast separation of proteins and larger biomolecules. The particles' 300-Å pore diameter was sufficiently wide to accommodate most proteins commonly encountered. Now, widepore SPPs are being reinvestigated by several research groups. At HPLC 2012, Kirkland and co-workers reported on their work on SPPs with larger pore sizes than the current 90 Å used for small-molecule separations. They investigated a number of parameters that may affect the separation of larger molecules, such as particle size, pore size, shell thickness and ligand length. Three shell particles were examined: 2.7μm core with a shell thickness of 0.35 μm; 2.7-μm core with a shell thickness of 0.2-μm; and 3.4-μm core with a shell thickness of 0.2 μm. Using ribonuclease (13.7 kDa) as a test solute, van Deemter curves showed that the larger particle provides lower 

 values than the smaller particles did. Presumably, the larger particle size packed more efficiently in the column. Different pore sizes were examined: 90, 160 and 400 Å. The smallest pore size showed restricted access for solutes with molecular sizes of 5000 Da and higher. The intermediate pore size could accommodate molecules as large as 15000 Da before peak shape was affected but the largest pore size showed no restriction for most proteins. The 2.7-μm, 400-Å particle gave better efficiencies than a totally porous 300-Å particle when tested under the same conditions. They also investigated alkyl chain lengths of bonded phases and found that C4 and C8 gave superior performance compared to C18. A rabbit skeletal myosin sample gave better resolution of light chain (17 KDa) and heavy chain (220 KDa) monoclonal antibodies on the 400-Å, 2.7-μm particles than on the 160-Å, 2.7-μm particles.  

Monolith columns have been desirable for a long time because they exhibit high permeability and low pressure drop (because of the increased bed porosity), show reasonable separation efficiencies, have the absence of frits to confine the packing material, are easy to fabricate, and can be made fairly reproducibly. Although this technology has been around for several years, as a routine tool it has yet to see widespread acceptance on the commercial side; however, improvements continue to be made for both polymer- and silica-based monoliths. HPLC 2012 had three oral sessions (11 talks), one tutorial and 31 posters devoted to the monolith technology. As mentioned earlier, reports on polymeric monoliths outnumbered silica monoliths by a 2.5:1 margin. 

In his plenary lecture on the first full day of the symposium, Nobuo Tanaka of GL Sciences and the Kyoto Institute of Technology in Japan gave an excellent overview of the status of monolithic silica columns for HPLC. Monolithic structures allow independent control of through-pore size and skeleton size providing the possibility for higher performance than that of particulate columns. He provided a practical comparison of TPP and SPP columns with first- and secon-dgeneration silica monoliths as depicted in Table 6. 

Table 6: Practical comparison of modern HPLC columns with silica monoliths. 

For small-diameter columns of 2 mm i.d., the performance of monolithic silica columns at 20 MPa or lower pressures can be compared favourably with that of a column packed with sub-3-μm core–shell particles or sub-2-μm TPPs operated at a much higher pressure. Such monolithic columns allow for the use of conventional 400-bar HPLC instrumentation.  

Column Watch

Highlights of HPLC 2012

, recently organized a panel discussion on current trends in high performance liquid chromatography (HPLC) instrumentation and how the technology could develop in the future. 

A panel of experts (listed in sidebar) were asked to discuss the most important developments in HPLC instrumentation over the past two decades. Ultrahigh-pressure liquid chromatography (UHPLC) — with its small-particulate columns, ultralow-dispersion fluidics, low dead volumes and ability to achieve fast separations — topped the list. 

"In many industries, UHPLC has opened up new approaches to working practices in terms of speed of analysis and ability to better 'fingerprint' samples," said Paul Ferguson of The Chromatographic Society in the UK. 

Richard A. Henry of the Pennsylvania State University agrees that UHPLC was a significant development, but sees important downsides to it. "The current benefits of UHPLC instruments may be offset by reliability issues, high instrument pressure drop, loss of temperature control, and more," he says. "And the cost of doing HPLC has gone up very dramatically." 

Mansoor Saeed of Syngenta feels the most important developments were two of the enabling technologies for UHPLC. One is the design of pumps that made it possible to increase operating pressures to 15000 psi, reduce dwell volumes to below 45 µL and improve solvent blending to refine gradient operations. The other was increasing detector sampling rates from 10 Hz to more than 100 Hz. "This has led to the expansion of eleven different instruments from ten manufacturers in the HPLC and UHPLC segment, each with a different power range," he says. 

Mass spectrometry (MS) detection (with the introduction of electrospray ionization, ESI), was cited as another key development that greatly expanded the applications of LC, especially in the life sciences. Many pointed to not just the sensitivity of LC–MS but also its current ease of use, which has made it a standard analytical tool.  

"The availability of effective, commercially available and reliable LC–MS instruments that can be used routinely by chromatographers rather than mass spectrometrists was important," said Bob McDowall of McDowall Consulting. "These instruments provide selectivity and sensitivity, making them instruments of choice in areas where these parameters are required." 

In particular, atmospheric-pressure ionization techniques were also named several times. "While not quite a universal detector, atmospheric-pressure MS has made it possible to measure compounds at increasingly low levels with relatively greater ease," says Howard Hill of Huntingdon Life Sciences. "It also provides the possibility of increased throughput, even though the latter has not always been exploited."  

Data processing and systems integration were high on the list for a number of our experts. Mel Euerby, of Hichrom Limited and Strathclyde University, particularly values the development of robust, automated method development platforms, including column- and solvent-switching technology coupled with triple detectors (UV, MS and charged aerosol detection, also called corona discharge detection [CAD]) and retention modeling software programs. 

Mark Argentine of Eli Lilly and Company said the development of highly flexible and precise variable volume, programmable autosamplers approximately two decades ago had a tremendous impact on laboratory efficiency and analytical data quality. "While we take such technology for granted in most analytical equipment today, tools such as these have enabled analysis of precious (microvolume) samples in a wide variety of applications,including high-throughput screening and analysis of biological materials," he said.  

Ferguson feels the introduction of reagent-free ion chromatography (IC) was a key development. "This instrument, co-developed by Paul Haddad's group at the University of Tasmania and Dionex, has greatly simplified the way analysts can perform IC, and importantly, transfer these methods across organizations," he said.  

Panel co-chair Tony Fell, of the University of Bradford, stressed the important achievement of chiral separations — for chiral analytes and biological targets, with diverse reversed-phase and normal-phase packings coupled with sensitive MS detection.  

Fell also noted an important underdeveloped area. "The lack of sensitive detectors based on optical activity — such as circular dichroism and polarimetry — is a major disappointment," he says. 

Clearly MS detection has been a critical recent development. So what do our panelists see for the future of detection technology?  

"Mass spectrometry increasingly will become a standard detector for any lab environment, from research down to QA/QC," predicts Wim Decrop of Thermo Fisher Scientific. "MS will continue to improve in terms of ease of operation, data acquisition times, sensitivity, and resolving power."  

"Detector improvements will still revolve around MS, such as advances in high-resolution MS and the ability to rapidly and automatically optimize these systems, enhancing their appeal," says Hill. But for these developments to have maximum impact, he says, parallel developments in software, computer storage, and data accessibility will be essential. 

Both Euerby and Ferguson expect to see smaller footprints and greater portability for LC–MS instruments. That, adds Euerby, should also lead to the development of more affordable, pared-down mass spectrometers for certain applications — such as single-quadrupole MS systems for fast scanning. 

Progress is also anticipated with other types of detection, particularly evaporative light scattering detection (ELSD) — useful for large molecules — and CAD coupled with flow-through detectors to enhance sensitivity and identification with appropriate solutes and mobile phases. Panelists see the potential for increased scan speeds, reduced dispersion and better linear responses with these instruments.  

Fell recalls that since his prediction of UV diode-array detection (DAD) as the standard LC detector some 30 years ago, this detection method continues to make a major impact. "In the past two decades, detection has improved the most with diod-earray UV spectrometers and MS, making major contributions to lowering detection limits and providing solute identification," noted Henry. 

Gordon Marr of Patheon, a pharmaceutical contract research and manufacturing organization, sees a need for more development in in-line chiral detection technology, as noted above by Fell. "This technology has been available for more than 20 years, but because it is treated like a premium market, little progress has been made," he says.  

Decrop, for his part, foresees that electrochemical detection will become more compatible with UHPLC. "By improving ease-of-use and integration into LC systems, multichannel electrochemical detection has a great potential to become a standard LC detector," he says.  

But the quest continues for the holy grail of a universal detector — one that will respond equally to all analytes regardless of physicochemical properties.  

"Such a detector would be able to provide a relative measurement in metabolic routes, particularly where no synthetic standard is available, and avoid the requirement for a radiolabeled analogue," anticipates Saeed. "The detector technology would require properties similar to chemiluminescent nitrogen and corona aerosol detectors but with high dynamic range." One possible way to get there, he continues, would be to use novel ionization sources in LC–MS that can impart ionization to components of interest and reduce matrix effects.  

Ferguson believes the path to a universal detector may be through the development of flame ionization detection (FID) compatible with fluidic chromatography.  

Fell, however, foresees another route to universality. "Perhaps this will be based on ELSD or CAD, but only when the technical issues with response linearity, dead-volume, and reliability have been resolved." 

The panel showed broad agreement that MS detection was a critical development, and that this and other detection methods will continue to develop. But what is the current status of hyphenated LC techniques? 

Saeed notes that dramatic advances — in sensitivity, ease of use, the wide application to the analysis of large and small molecules, as well as the development of suitable software for identification of proteins and metabolites — have greatly expanded the use of hyphenated technologies, particularly those that couple chromatography to high-resolution accurate mass spectrometry. The major bottleneck, he says, is data generation, increasing the demand for automated data processing software. 

In bioanalysis, says Hill, all MS technologies play a major role and are spilling over to the formulation analysis field, where they are finding a role as a replacement for DAD. "This is certainly true in the method development lab, if not yet in routine analysis," he says.  

LC–MS is a mature technique and has migrated from an expert system to an open-access, walk-up format in many laboratories, says Argentine. He also notes that software advances have greatly advanced the productivity and utility of HPLC–MS systems for routine use in the analytical laboratory. "These tools afford opportunity for advanced analyses of extremely complex sample matrices, including biological samples," he says.  

A panel of experts give their opinions about the current trends in high performance liquid chromatography (HPLC) instrumentation and how the technology could develop in the future.

LCGC Europe-09-01-2012

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Highlights of HPLC 2012

Liquid Chromatography Instrumentation: The State of the Art

A Short Introduction to Multidimensional GC

Readers' Questions