Bioanalytical Tools for the Characterization of Biologics and Biosimilars 

In spite of the recent slowdown in the approval rate of new biopharmaceutical entities, dozens of new biologicals were introduced in the United States and the European Union during the past 5 years, adding up to more than 200 products already on the market (1). One of the fastest growing segments in this market is biosimilars (also referred to as follow-on biologics or subsequent entry biologics). This emerging biotech sector requires novel bioanalytical tools to address the challenging regulatory aspects (2), which entail comprehensive characterization by sensitive and high resolution bioanalytical methods covering all four structural levels of biotherapeutic proteins (that is, from primary to quaternary). During the development phase of both the novel biologics and biosimilars, full sequence coverage, purity assessment, quantification, and product identification is necessary. In addition, quite a few other critical features should be analyzed, such as glycosylation (including their microheterogeneity and site specificity), mutation, phosphorylation, sulfation, disulfide linkages, oxidation, deamidation, glycation, proteolytic clipping, and several others. Analysis of isomerization, especially isoaspartic acid formation (isoAsp and isoD), is another important requirement for their analysis because it might reveal immunogenic structural changes (3). Identification of host-cell impurities, aggregation, and determination of higher order structures and isoforms are also highly important (4).  

The analytical needs depend on the actual application in hand; for example, high throughput is a prerequisite during clone selection and high sensitivity is important during product release. To attain such levels of characterization, orthogonal bioanalytical methods should be applied including liquid chromatography (LC), capillary electrophoresis (CE), mass spectrometry (MS), and their combinations: LC–MS and CE–MS. These methods enable thorough analysis of intact proteins (top down), peptide digests (bottom up), and even in between (middle down or up) (5). The most frequently used proteolytic enzyme for these applications is trypsin that produces the majority of fragments below 3 kDa, a size range that is readily compatible with most present day MS instruments. Higher resolution mass spectrometers such as hybrid MS units enable routine analysis of larger fragments with higher charge states (6).  

Mass spectrometers are the most important workhorses in the characterization of biologics and biosimilars (7,8). Important features include mass resolution and accuracy. In this respect, Table I delineates the capabilities of the top-of-the-line MS systems these days.  

However, even for the best mass spectrometers on the market, the use of separation methods (LC or CE) before the mass analysis is highly recommended to decrease the complexity of the sample entering the device (9), as shown in Figure 1. Pre-MS separation also increases the dynamic range and decreases ion suppression in mass spectrometry. One of the preferred separation modes is LC, typically using narrow-bore or capillary columns with packing particle sizes of 3–5 µm or lower (1.7 µm). The porosity of the stationary phase should match the size of the solute molecules; for example, for peptides it should be 20 nm and for proteins 30 nm. The pressure drop on such systems ranges from 6000 to 15,000 psi depending on the particle size and the column length. Ultrahigh-pressure liquid chromatography (UHPLC) provides higher resolution and sensitivity for peptide mapping than regular high performance liquid chromatography (HPLC), enabling better sequence coverage and variant characterization (10). Other separation parameters, such as column temperature, are also important for special applications. Because of its higher speed of analysis, UHPLC is a good choice for high-throughput peptide mapping, providing fine details about differences between a biosimilar and its innovator product counterpart. State-of-the-art software tools help with automated searches of LC–MS datasets to assign peptides and annotate peptide variants in minutes even at low levels, a process that manually takes several days per data file. Differential plotting options enable quantitative comparison between different peptide maps of, for example, a biosimilar to an innovator product. 

Capillary electrophoresis is another high efficiency separation method frequently used in bioanalysis providing fast separation (minutes to seconds) with predictable selectivity, requiring only small sample volumes (1–10 µL) (11). Features of CE also include quantification in a reasonable dynamic range. The option to use multicapillary devices can significantly increase the throughput of the analysis (for example, DNA sequencing). Coupling CE to a mass spectrometer is an emerging field of research. From the various modes of CE, capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), and capillary isoelectric focusing (cIEF) are most frequently used for the analysis of biologics and biosimilars. 

In the analysis of biotherapeutics, with the goal of good recovery, the hydrophobicity and size of the sample molecules should be considered as well as their aggregation tendency. Samples should be treated accordingly with detergents or denaturants. In peptide analysis by LC–MS, the size and hydrophobic or hydrophilic characteristics of the peptides should be considered. If the peptides are too small or hydrophilic then they are not retained. If the peptides are too big or hydrophobic then they are not recovered properly. To avoid these size-related issues, one option is to implement a multienzyme approach that provides suitable sized peptides for the analysis, keeping in mind that peptide sizes between 1000 and 3000 Da work well for most LC–MS systems. On-line LC–MS, for the analysis of larger peptides, may need a combination of collision-induced dissociation (CID) and electron-transfer dissociation (ETD). CID of an isolated charge-reduced species derived from ETD is an effective approach to determine phosphorylation sites and glycosylation modifications as shown in Figure 2 (12). To further analyze glycosylation modifications, hydrophilic interaction liquid chromatography (HILIC) stationary phases have been introduced using amide, diol, amine, aminopropyl, and zwitterionic (ZIC-HILIC) phases (13). However, normal-phase silica-based columns are still being used for glycopeptide or glycan analysis. Capillary electrophoresis (as well as CE–MS) is another good option to analyze glycosylated proteins or peptides (14). Rapid analysis of glycosylation, especially for immunoglobulins, is also possible by a novel microchip-based LC–MS approach that utilizes an integrated microfluidics-based sample preparation system before MS analysis (15). The built-in PNGase F enzyme reactor cuts off the IgG glycans, and the resulting glycosylamines are separated on a porous graphitized carbon column followed by detection in the connected MS unit. 

Effective sample preparation for the analysis and characterization of biologics and biosimilars is a key issue (16). This includes the collection and handling of the samples as well as the reproducibility of process protocols. Introduction of quality control (QC) points is highly important to monitor the process. Also, reagent components along with their potential effects on the sample during downstream processes (for example, detergents, denaturants, extreme pHs, and protein concentration) should be considered. System suitability tests are necessary to ensure the performance of the analytical approach. Thus, it is important that the sample preparation process is reproducible, including solubilization of all proteins of interest. Protein aggregation should be avoided during sample preparation and the concomitant analytical processes. Also, chemical modifications of the proteins or peptides should be prevented and interfering molecules should be removed. This is important during clone selection and stability studies, in which detection of undesired protein forms are crucial. Obtaining proteins of interest at a detectable level may involve the removal of abundant or nonrelevant classes of proteins. If digestion is among the sample preparation steps, its reproducibility and efficiency is also very important. 

Among the important critical features, analysis of post translational modifications, especially glycosylation, is becoming more and more important during the characterization of biotherapeutics. The typical goal is to decipher the heterogeneity of the attached glycans and recognize the multiple glycosylation sites per protein. As a matter of fact, each site can hold multiple glycan structures resulting in hundreds of glycoforms, which might be responsible for various biological activities (17). Many factors contribute to alterations in glycan processing during the production of therapeutic glycoproteins, thus their analysis during the process is crucial. With complicated samples such as complex glycan mixtures, the use of orthogonal separation methods is a key to provide full characterization. CE and CE–MS analysis of intact glycoproteins (18) or the released glycans (19) can achieve such a goal. Apparently, reversed-phase LC–based protein separation has limited resolving power for glycoforms. CE, however, in which separation is primarily based on charge and size, provides high resolving power for glycoforms and is helpful in the characterization of multiple glycosylation sites (14).  

The comprehensive analysis of intact glycoproteins can be done by first separating them using chromatography or electric field–mediated techniques. Ion-exchange chromatography has limited resolving power, but isoelectric focusing provides high resolution in charge-based separation. Before the separation and analysis, appropriately gentle sample preparation steps such as size-exclusion chromatography or molecular weight cut-off filtration are necessary. Among the MS-friendly separation tools, reversed-phase HPLC has only limited resolution for glycoforms. CE separates analyte molecules based on their charge, mass, and molecular shape; however, it requires MS-compatible buffer systems and minimal dead volume at the electrospray ionization (ESI) interface. Typical approaches include using sheath flow, sheathless, or liquid junction methods (20). The orthogonality of the above listed separation methods should be fully utilized in the analysis of biotherapeutics, both for innovative biologics and biosimilars. It is especially important during the development phase as glycosylation processing (microheterogeneity and site specificity) is very host-cell dependent and sensitive to cell culture conditions.  

During MS based assignment of glycopeptides, the peptide backbone sequence is obtained by ETD after selecting the best candidates from the CID fragmentation. Confirmation can be accomplished by accurate mass determination or with orthogonal analytical methods such as CE, high-pH anion-exchange chromatography, or reversed-phase LC. Analysis of the released glycans is also possible by MS. However, simple MS can provide information only on glycan composition with respect to the number of hexoses (for example, galactose and mannose), 

-acetyl-hexosamines (GlcNAc and GalNAc) as well as sialic acids (Neu5Ac and Neu5Gc), and provides no information on their linkages or positions (21). By using MS-MS with appropriate fragmentation modes (CID, post-source decay [PSD], ETD, or photodissociation) some linkage information can be obtained. This, again, emphasizes the necessity of a separation dimension before MS detection and analysis. With the use of MS-friendly high-resolution separation techniques, positional and linkage isomers can be separated and consequently analyzed. The HPLC separation of native glycans can be accomplished by porous graphitized carbon stationary phases, and derivatized glycans (2AB or 2AA) are better separated using HILIC separation mode. The charge-based separation by high performance anion exchange chromatography (HPAEC) is a good tool for determining characteristics such as the number of sialic acids. Another important separation tool before MS analysis is CE of highly charged fluorophore (aminopyrene trisulfonate [APTS] or 8-aminonaphthalene-trisulfonate [ANTS]) labeled glycans. Figure 3 compares the separation of IgG N-glycans by HILIC and CE–laser-induced fluorescence (LIF) methods. An important issue is the localization of the actual glycosylation sites, and because peptides with different glycoforms are eluted closely, MS-based verification is used to obtain such information.  

One of the most frequently used applications of CE in the analysis of biotherapeutics and biosimilars is the characterization of protein glycosylation, which requires preseparation derivatization with a charged fluorophore after the release of glycans (both N- and O-linked). This reductive amination-based, rapid, single-step labeling reaction (usually with APTS) features great efficiency (>90%) and provides a triple-charged fluorescent tag supporting excitation at 488 nm with 520-nm emission. Quantification is easy because only one fluorophore is conjugated to one sugar entity (23). Figure 4 delineates a CE-based glycan analysis workflow for therapeutic antibodies, starting with sample purification with protein A cartridges, through APTS labeling, sample cleanup, CE analysis, and database query. Such characterization of monoclonal antibody (mAb) N-linked glycans readily gives comprehensive information on their structural diversities, such as the presence of core fucosylation or immunogenic sugar residues, such as alpha 1→3 galactosylation or 

-glycolylneuraminic acid. This assessment of glycosylation microheterogeneity is very important during clone selection, process control, and lot release for both the therapeutic recombinant mAbs and their biosimilar counterparts. 

Disulfide mapping is another important characterization method, which requires treatment with multiple enzymes and multimode MS to reveal disulfide-linked sites and the sequence information of the linked peptides (24). ETD cleaves the disulfide bonds preferentially over the peptide backbone and CID cleaves the peptide backbone while leaving disulfide bonds intact. The specificity of enzymes used for disulfide mapping should be high enough to avoid miscleavages when multiple disulfide links are in close proximity. Another issue to consider is that enzyme digestion often fails to cleave a desired amino acid right next to a disulfide bond (25). Glycosylation in disulfide-linked peptides further complicates their analysis as the efficiency of peptide N glycanase F (PNGase F) can be poor or the peptide may not be fully recovered after deglycosylation, both leading to decreased sensitivity. Please note that when using ETD fragmentation, the disulfide bonds are preferentially cleaved over glycosidic bonds. Disulfide assignment by ETD or CID uses precursor masses to obtain the most likely disulfide-linked peptides. ETD obtains disulfide-dissociated or partially dissociated peptides, which can be confirmed by further fragmentation with CID. 

Even though most of the analyses of biologics and biosimilars focus on the primary structure, the higher order structures of these molecules is also highly relevant, and they are invisible to the above described techniques. Several classical biophysical tools can be used for global structural analysis of biologics and biosimilars, such as analytical ultracentrifugation, size-exclusion chromatography, fluorescence, circular dichroism, isothermal titration calorimetry, and differential scanning calorimetry. These techniques can reveal information about secondary, tertiary, and quaternary structures, but currently there is still a lack of full understanding about the conformations of most therapeutic proteins.  

Hydrogen or deuterium exchange using mass spectrometry (HX-MS) is another valuable method for protein conformation analysis because the kinetics of H–D exchange is highly dependent on the protein conformation (26). Advantages of HX-MS include relatively good resolution, picomole-range sample requirement, no restriction in protein size, and a relatively high tolerance of sample impurities. The approach provides data on conformational changes during function and helps identify interactions or surfaces with the aid of known structures. Alternatively, it also provides some useful information when the structure is unknown. Besides being a valuable tool for studying protein function, HX-MS is emerging as a method of quality control in biopharmaceutical development and a diagnostic tool for proper folding. 

Biotherapeutics are highly complex molecules with complicated higher order structures that are subject to a plethora of post-translational modifications. The biologic substance (the molecule itself) and biologic product (the pharmaceutically formulated final product) also contain process- and product-related impurities such as aggregates, heterogeneous structures, and fragments (27). These factors underline the importance of the subject matter discussed in this column, helping to understand the analytical science accompanying the development of these complex therapeutic molecules and keeping pace with the rapidly developing regulatory framework for these products. 

http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm122858.pdf

(3) H.A. Doyle, R.J. Gee, and M.J. Mamula,  

(4) J. Carpenter, B. Cherney, A. Lubinecki, S. Ma, E. Marszal, A. Mire-Sluis, T. Nikolai, J. Novak, J. Ragheb, and J. Simak, 

(6) X. Han, A. Aslanian, and J.R. Yates, 3rd, 

(10) U.D. Neue, M. Kele, B. Bunner, A. Kromidas, T. Dourdeville, J.R. Mazzeo, E.S. Grumbach, S. Serpa, T.E. Wheat, P. Hong, and M. Gilar, 

(11) B.L. Karger, A.S. Cohen, and A.J. Guttman, 

(13) G. Zauner, A.M. Deelder, and M. Wuhrer,  

(14) D. Thakur, T. Rejtar, B.L. Karger, N.J. Washburn, C.J. Bosques, N.S. Gunay, Z. Shriver, and G. Venkataraman, 

(17) A. Varki, R. Cummings, J. Esko, H. Freeze, P. Stanley, C.R. Bertozzi, G.W. Hart, and M.E. Etzler, 

Essentials of Glycobiology, Second Edition

, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2009). 

(18) R. Haselberg, G.J. de Jong, and G.W. Somsen, 

(22) S. Mittermayr, J. Bones, M. Doherty, A. Guttman, and P.M. Rudd,

(23) Z. Szabo, A. Guttman, T. Rejtar, and B.L. Karger, 

Articles

Biotechnology Today

LC–MS and CE–MS are the go-to techniques for characterizing biologics, but often help is needed from a range of other methods.

Bioanalytical Tools for the Characterization of Biologics and Biosimilars

There are many factors influencing the efficiency of a high performance liquid chromatography (HPLC) stationary phase. Of these factors, chemical nature of the bonded phase ligand is important, but by no means all encompassing in determining the important phase characteristics. 

In 2005, there were about 220 C18 (L1) phases available (1). One can only speculate on the number available today, and each will be subtly different in separating the analytes that we are interested in. 

As users and developers of HPLC methods, we need to improve our understanding of the factors affecting separations so that we can better understand problems when they occur and learn to exploit key stationary-phase characteristics to our advantage during method development or improvement. 

This might be achieved by testing each (and every!) stationary phase using a standard set of chemical probes that we know will react in a predictable way, depending upon the phase characteristics. In this way, we can produce comparative data that will allow us to select phases that we suspect might be best at exploiting important chemical and physicochemical differences between our analytes. We can then map column characteristics and group columns (even those of the same "nominal" bonded phase) into those that are "similar" or "different" (sometimes called "orthogonal" in this context), allowing us to manipulate our analyte retention and separation selectivity accordingly.  

Several attempts have been made to produce a definitive set of chemical probes and tests to best characterize the huge number of stationary phases available (currently estimated to be well over 1000 distinct chemistries). As yet, a harmonized set of test probes and methodologies has not come to the forefront; however, there are some notable groups of scientists working in this field. 

An early attempt at producing a generic set of probes for testing HPLC column characteristics was made by Tanaka and coworkers (2). Since then, work by the USP Working Group on HPLC Columns, the Impurities Working Group of the PQRI Drug Substance Technical Committee in collaboration with Lloyd Snyder (3), and Euerby and Petersson (4) has expanded the original probes designed by Tanaka. These groups have all attempted to identify a definitive set of probes that will allow the various important physicochemical phase characteristics to be specified. Most of these researchers also have combined their data with various chemometric and statistical approaches to produce quantitative databases based on principal component analysis (PCA) or other statistical methods to visualize the relative groupings of commercially available columns according to their key descriptors as well as computing a single numerical "similarity" factor, for simple comparison of orthogonality.   

Even though the chemical probes used differ between the research groups, there are some common themes in the attributes of a stationary phase that are considered of primary importance in characterizing the stationary phase behavior:  

ability to discriminate between probes of similar hydrophobicity (hydrophobic selectivity)

ability to discriminate between analytes of different shape or hydrodynamic volume (shape or steric selectivity)

extent of hydrogen bonding with acids or bases (typically via the silanol surface, polar end capping reagents, or functional groups within the bonded ligand)

extent of ion-exchange interactions at low and mid pH (pH 2.8 and 7.0 is typical to differentiate between situations in which surface silanol species will be potentially ionized or ion suppressed).

Figure 1 shows how one might simply represent column characteristics for comparison.  

Figure 1: Spider diagram representing the various characteristics of a stationary phase (Accucore PF, Thermo Fisher Scientific). HR = hydrophobic retention; HS = hydrophobic selectivity; SS = steric selectivity; HBC = hydrogen bonding capacity; BA = base activity; C = chelation; IEX = ion-exchange capacity at pH 2.6 and 7.6; AI = acid interaction. (Reproduced with permission from Thermo Fisher Scientific.) 

The various databases developed by these three groups can be used to identify similar or different phases and, with some increased understanding of the various characteristics described, make some rudimentary predictions about which column (type) might be best suited for a particular analysis. 

The three main databases are all publicly available and can be found at the following locations:  

ACD Labs Column Selector — based on the work of Euerby and Petersson: 

www.acdlabs.com/products/adh/chrom/chromproc/index.php#colsel

(2) K. Kimata, K. Iwaguchi, S. Onishi, K. Jinno, R. Eksteen, K. Hosoya, M. Arki, and N. Tanaka, 

(3) N.S. Wilson, M.D. Nelson, J.W. Dolan, L.R. Snyder, R.G. Wolcott, and P.W. Carr, 

The Essentials

Can databases of column characteristics be used to help with HPLC column selection ahead of method development?

Column Characterization Databases

Part II of our yearly report on new products introduced at Pittcon. This month, gas chromatography columns, thin-layer chromatography plates, sample preparation products, as well as chromatography and sample preparation accessories (including small benchtop instruments) are covered.

Pittcon 2012 (more formally known as the 63rd Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy) returned to the Orange County Convention Center in Orlando, Florida, March 11–15, 2012, after a one year absence. This year's event hosted 948 instrument manufacturers and laboratory suppliers in 1854 booths. In addition to attending the exposition, conferees listened to more than 2000 technical presentations (orals, posters, workshops, invited and contributed talks, and award symposia), checked on numerous company seminar rooms, or attended one of 95 short courses. 

Although the average attendance has been shrinking over the last several years, undoubtedly, Pittcon still remains the most important yearly international analytical exhibition where companies introduce their latest instruments, instrument accessories, software, columns, sample preparation, and other consumable products. Because many past attendees have purchased one or more new products within three months after attending the show, most exhibitors attempt to maximize their booth traffic to meet as many potential customers as possible. 

The purpose of this report is to provide information about many of the new separation consumables and accessory products that were displayed at Pittcon 2012. In some cases, products that were introduced during 2011 but after Pittcon 2011 (1,2) may be included for reasons of completeness. The information is based on manufacturers' responses to a questionnaire mailed in early 2012. Because of space limitations and the fact that some manufacturers did not respond to the questionnaire, this report cannot be considered an exhaustive listing of all new products that were introduced since last year's event in Atlanta. However, over the years, these Pittcon introduction summaries have provided a good source of information that would be difficult for one individual to gather during the four days of the exhibition. In addition, the products introduced have shown definite correlations to current research, development, and application activity in the separation sciences. 

As in previous years, products recommended by their manufacturers primarily for biomolecule and biofluids sample preparation are denoted in the tables with the designation 

. Some of these products may be used for general sample prep as well, but their main emphasis is for biological samples.  

In this month's coverage, I will look at gas chromatography (GC) columns, thin-layer chromatography (TLC), sample preparation products and hardware, accessories, and small tabletop instruments, mainly for sample preparation. 

Although GC is considered a relatively mature separation technique, new GC columns continue to be introduced each year at Pittcon (Table I). This year, we saw the introduction of eight new GC columns: seven wall-coated open tubular (WCOT) columns and one porous layer open tubular (PLOT) column. Columns were introduced by six different vendors. In addition, Agilent Technologies introduced an entire line of J&W packed columns for laboratories still using this technology. These columns are still accepted in many environmental and pharmaceutical applications. 

Application-specific GC columns are always popular introductions at Pittcon. This year, a majority of the columns were for specific application areas. GC has found widespread usage in the petroleum industry. A single PLOT column was introduced by Restek for the trace-level analysis of volatiles in hydrocarbon streams, particularly for downstream products such as ethylene, propylene, the butylenes, and butadiene. Supelco/Sigma Aldrich also introduced a hydrocarbon processing industry (HPI) column for the analysis of benzene and other aromatics in gasoline, but the proprietary ionic, very polar liquid stationary phase also can be used for fatty acid methyl ester (FAME) profiles in biodiesel and edible oils. Ionic liquids are a new breed of GC phases that have higher temperature limits than typical coated or bonded GC phases. To illustrate the capability of this new type of phase, Figure 1 shows the separation of a standard 37-component FAME mix (Supelco/Sigma Aldrich) containing methyl esters of fatty acids ranging from C

, including key monounsaturated and polyunsaturated fatty acids. Although not shown, Supelco's standard column for FAME analysis has been a cyanopropyl siloxane phase (SP-2560) and it was compared with the SLB-IL 111 ionic liquid phase column. The newer SLB-IL 111 column gave a slightly better signal-to-noise ratio, had a reduction in analysis time because of weaker dispersive interactions, and showed stronger dipole-induced dipole interactions resulting in increased retention of polarizable analytes (those with double bonds) relative to nonpolarizable analytes. The higher the degree of unsaturation, the stronger the retention; thus, an alternative selectivity is noted with the SLB-IL 111 column relative to the SP-2560 cyanopropyl siloxane column.  

The workhorse column in many GC and gas chromatography–mass spectrometry (GC–MS) laboratories is the 5% phenyl–95% polysiloxane phase. Three companies introduced a 5% phenyl column, but one of them has a polysilphenlene siloxane backbone that is said to increase the thermal stability and decrease column bleed. Also, Thermo Fisher Scientific's version was available in stainless steel metal configurations. 

TLC (and high performance TLC [HPTLC]) is a simple technique that many chromatographers overlook, even when they only need a cursory separation or purity check. Macherey-Nagel is one of the few companies that produces thin-layer products. They have expanded their Alugram Xtra line of TLC and HPTLC products to a wide variety of formats. The base product is unmodified standard silica layers on aluminum. The silica gel has a specific surface area of 500 m

 /g, a mean pore size of 60 Å, and a specific pore volume of 0.75 mL/g. The particle size is 5–17 µm. The binder holding the silica to the aluminum plate is a highly polymeric product so that the plate can be cut easily without flaking the silica. The layer is stable in almost all organic solvents including pure aqueous eluents. The HPTLC plate has a smaller particle size of 2–10 µm, which gives sharper separations, shorter developing times, and increased detection sensitivity compared to the regular TLC plates. The plates have an indicator of manganese-activated zinc silicate with green fluorescence of shortwave UV (254 nm). Special plates are available with a concentrating zone. 

As indicated in Table II, Pittcon 2012 was another productive year with 19 new sample preparation products or families of products displayed. Not included in that count were sample preparation accessories (Table III), which will be covered later. The most popular application areas include various pollutants in environmental samples, drugs and drug metabolites in biological fluids, and toxins in food. Formats included cartridges, 96-well plates, and spin columns for use with a centrifuge as the driving force.  

Many of the products were for solid-phase extraction (SPE) and just about every mode (reversed phase, strong or weak anion and cation exchange, adsorption and normal phase, partition, and mixed mode) was represented. A wide variety of media was introduced at Pittcon including polymer-based, graphitized carbon black, silica-based bonded phases, bare silica, alumina, titania, and diatomaceous earth.  

Table II: Sample preparation products (continued) 

The majority of the SPE products introduced at Pittcon 2012 were polymer-based.   

Polymeric SPE products are particularly attractive because they can dry out during the conditioning steps without affecting the recovery and reproducibility of the SPE experiment. Also, they can be treated with more aggressive washing solvents than silica-based sorbents because their pH range is greater. Generic methods are often used, which makes method development simpler. Another advantage of the polymeric SPE sorbents is that they usually display mixed mechanisms and multiple interactions can be used to selectively retain analytes or matrix compounds. Because the surface area of polymers is higher than that of silica-based bonded phases, a smaller amount is used in cartridges and well plates. Thus, smaller amounts of sample and solvents are required and extraction times and evaporation times are faster.  

For the third year in a row, additional products for QuEChERS were introduced. Pronounced "catchers," QuEChERS is an acronym for quick, easy, cheap, effective, rugged, and safe sample preparation. This method is an emerging technique that has become increasingly popular in the area of multiresidue pesticide analysis in food and agricultural products. In QuEChERS, food and agricultural samples are first extracted with an aqueous miscible solvent (for example, acetonitrile) in the presence of high amounts of salts (such as sodium chloride and magnesium sulfate) or buffering agents (such as citrate) to induce liquid-phase separation and stabilize acid- and base-labile pesticides, respectively. Upon shaking and centrifugation, an aliquot of the organic phase is subjected to further cleanup using SPE. Unlike traditional methods using SPE tubes, dispersive SPE (d-SPE) cleanup is conducted by mixing bulk amounts of SPE sorbent and additional MgSO

 with the extract. After sample cleanup, the mixture is centrifuged and the resulting supernatant can either be analyzed directly or subjected to minor further treatment before analysis. Two new QuEChERS product offerings were submitted for inclusion in this article (see Table II). For those interested, a brief introduction to the technique was published in earlier installments of "Sample Prep Perspectives" (4,5).  

Table III: Chromatography and sample preparation accessories 

The technique of dried blood spotting (DBS) has been drawing interest in the pharmaceutical industry for the sampling, storage, and shipping of blood samples for the analysis of drugs and metabolites (6). Basically, small volumes (~15 µL) of blood are spotted on a dried matrix card, the liquid is allowed to evaporate, spots are punched out of the media, and analytes of interest are eluted with solvent and either treated further or directly analyzed by liquid chromatography–tandem mass spectrometry (LC–MS-MS). Because more biofluids (for example, plasma, urine, and cerebrospinal fluid) than blood can be handled with DBS cards, it has been suggested that the technique be referred to as 

 (DMS). The cards are deemed safe as not being a biological hazard when blood and body fluids are dried. In addition, analytes are stabilized on the card when dried. The technique saves all the safety and logistics problems of storing and shipping whole blood samples. Products for DBS or DMS were shown at Pittcon, this time including new card formats (Table II) and an instrument that will desorb analytes on-line (Table III). 

Table III: Chromatography and sample preparation accessories (continued) 

The supported liquid extraction (SLE) technique uses small particles of diatomaceous earth packed into a cartridge. An aqueous sample (usually buffered) is applied to the packed cartridge and it distributes itself by capillary action over the sorbent. The high surface area of the sorbent ensures that the sample is finely dispersed. Next, an immiscible organic liquid is percolated through the cartridge and comes in intimate contact with the aqueous sample. A liquid–liquid extraction is performed, just as might occur in a separatory funnel using the two immiscible phases. Because vigorous shaking is not required, there is no chance of an emulsion forming. The organic liquid containing the analyte or analytes of interest is recovered from the cartridge outlet. The analytes can be obtained by evaporation. The entire process is very reminiscent of conventional liquid–liquid extraction, but is much simpler and is performed entirely on-line. New formats of SLE were introduced at the exhibition. 

Chromatography and Sample Preparation Accessories

Table III lists 42 new hardware products and accessories for HPLC, GC, capillary electrophoresis (CE), and sample preparation that were introduced at Pittcon 2012. This year, I have extended the columns and sample preparation accessories to include small, benchtop dedicated instruments that are designated to automate sample preparation techniques. All of the products introduced were practical devices designed to make a chromatographer's life easier. I will cover each area individually. 

 Several products that are useful for both HPLC or ultrahigh-pressure liquid chromatography (UHPLC) and GC (coupled to MS and MS-MS) were shown this year. Syringe filter configurations are used mostly in LC and occasionally in GC applications and three companies introduced new families of these devices. Thermo Fisher Scientific's Titan3, Target2, and Sterlin syringe filters have various membranes, porosities, and diameters to meet most needs. The Pall syringe filters are ultraclean and are recommended for LC–MS applications. The SGE Analytical syringes are also ultraclean for LC–MS and GC–MS applications. An interesting twist is that they have application-specific needles to cut down on analyte interactions, reduce possible sample loss, and minimize carryover.   

In this category, 96-well filtration plates are mostly used for HPLC or UHPLC applications but serve the same purpose as the syringe filters by removing particulates from samples. Filtration plates and spin columns now have established a place in the sample preparation laboratory. Many pharmaceutical companies now use protein crashing rather than SPE for the analysis of drugs and their metabolites in biological fluids, mostly using LC–MS-MS. Acetonitrile or methanol is added to diluted plasma and protein is precipitated, usually in a bead form. The supernatant is filtered and analyzed by LC–MS-MS. Because this type of testing is usually performed on many samples, 96-well filtration plates are the preferred format. Spin columns also perform rapid mechanical filtration, chemical filtration (by the use of adsorbents or molecular weight cut-off membranes), and desalting for small volumes of sample and are convenient when only a few samples need to be prepared. Several companies displayed new spin column formats and 96-well filtration plates. 

HPLC, UHPLC, LC–MS, and ion chromatography products:

 The advent of UHPLC has spurred a number of ultrahigh pressure products. This year, two companies introduced reusuable high-pressure fittings that will go as high as 27,000 psi. A high-pressure injection valve designed for pressures as high as 20,000 psi was shown. Guard columns are always a good idea and two companies introduced high-pressure guard columns, both rated to 20,000 psi, one of which can be vented using a switching valve. The popular sub-2-µm UHPLC columns

are particularly susceptible to plugging because of the small porosity frits, so guard columns or in-line filters are almost a must. A safety bottle cap that prevents loss of volatile solvents from solvent reservoir bottles, yet allows air to enter to prevent pump cavitation is a simple but practical device (Figure 2). For anybody who has used trifluoroacetic acid in his or her mobile phase only to find retention times changing because of trifluoroacetic acid evaporation will appreciate such devices. 

LC–MS-MS systems often use a lot of nitrogen in the 10 L/min range and higher. It is highly impractical to use nitrogen cylinders to provide these high flow rates. Two companies have come out with nitrogen generator systems for LC–MS-MS that generate flows as high as 60 L/min at pressures required for operation. 

 This year, only three product offerings strictly for GC were provided. An improved gas-tight syringe for headspace sampling was shown. The syringe features a spring-in plunger that made a tight seal against the glass barrel. Gas-tight connections are always a concern in GC flow systems. A new inert microconnector with low thermal mass for rapid temperature response was introduced. A line of ultra-inert inlet liners was extended by the introduction of a dimpled liner (for cold splitless injection) and a double-taper liner (vapor confined to inside of liner) as indicated in Table III. 

Sample preparation usually is the most popular area for new accessories, and this year proved to be no exception, with some clever products presented at Pittcon 2012. Almost three-fourths of all new products covered in Table III fit into this area. Small benchtop instruments that automate some of the sample preparation steps can save time, eliminate tedium, and reduce errors. Here, I cover 11 of these benchtop instruments that range from workbench designs that perform multiple sample preparation procedures on small sample volumes to a single task of solvent evaporation. Several of the instruments are capable of automating at least part of the SPE experiment. Two instruments are capable of assisting in pressurized solvent extraction. Shaking centrifuge tubes in QuEChERS is a tedious job, and one instrument automates this process.  

The combination of a precision delivery pipetting device and a miniaturized SPE needle is an interesting twist to the SPE experiment of small volumes. As depicted in Figure 3, the eVol-MEPS system from SGE combines the two devices to make a semiautomatic liquid handling–SPE device that performs all steps in the SPE experiment: conditioning, sample loading, washing, and elution. Because the mass of SPE packing is only a few milligrams and the volumes are a few microliters, small-volume cleanup of liquid samples is possible.  

In addition to the DBS cards introduced in Table II, two products for dried blood spotting were shown (Table III). Hand punching of dried blood spots is time consuming and tedious. One product shown at Pittcon was a foot-controlled card punch that will directly insert a punched disk into a 96-well plate (Figure 4); the second product is an automated system that will process as many as 500 DBS cards by providing on-line solvent extraction directly integrated with LC–MS.  

Almost anyone who does chromatographic samples uses 2-mL sample vials. Earlier, irregularly shaped vials were often the culprit when shutdown occurred on automated overnight runs or when trace analytes with sensitive compounds were lost. Recently, certified vials that are tested to rigid standards and LC–MS vials with low levels of extractables and inert surfaces have been introduced. At this year's Pittcon, additional high quality and certified vials were introduced by four companies. 

I would like to thank the manufacturers and distributors that kindly furnished the requested information in advance of Pittcon 2012, thus allowing a timely report on new product introductions. Manufacturers who would like to be considered for inclusion into Pittcon 2013 coverage should send the name of the primary company contact, the mailing address, phone number and e-mail address to Laura Bush, Editor, 

, with the subject line, "Pittcon 2013 Column Watch." 

(5) S.J. Lehotay, M. Anastassiades, and R.E. Majors, 

Column Watch

What you need to know about gas chromatography columns, thin-layer chromatography plates, sample preparation products, as well as chromatography and sample preparation accessories (including small benchtop instruments) unveiled at Pittcon this year

New Chromatography Columns and Accessories at Pittcon 2012, Part II

A dedicated gas chromatography–mass spectrometry (GC–MS) analyzer was developed to address the increasing need for more sensitive catalyst poisoner analysis. The system combines the separation power and robustness of a classic backflush configuration with the selectivity and sensitivity of MS.

The use of high-yield metallocene catalysts has dramatically increased both efficiency and selectivity of polymerization processes (1). Unfortunately, these catalysts are extremely prone to poisoning by feedstock impurities, such as arsine (AsH

), oxygenates (for example, dimethyl ether), and sulfur-containing compounds (mercaptans, sulfides, and so forth) (2,3). Minute amounts of these compounds are sufficient to impose undesirable effects and induce immediate loss of catalytic activity and reaction yield. At the same time, trace contaminants at parts-per-billion concentration levels can end up in the polymers and alter subsequent polymer properties and characteristics. 

For decades, process chemical and petrochemical analysts addressed their analytical challenges mainly by relying on superior chromatography and smart tools such as valve switching, backflush, and Deans heart-cut. In combination with relatively cheap, robust, and selective detectors, they were capable of providing all information necessary to control and tweak petrochemical processes. 

Organic catalyst poisoners are usually determined using dedicated chromatographic analyzers. These systems are, typically, equipped with a dual capillary column configuration with backflush and fitted with flame ionization detection (FID). Under these conditions, limits of detection are usually approximately 100 ppb, depending on the compound investigated and the complexity of the matrix that is introduced (4). Unfortunately, this is far from sufficient to protect the latest catalysts, which start to deteriorate as soon as fed with low parts-per-billion amounts (5,6). An overview of some typical specifications for catalyst poisoners in polymer-grade hydrocarbons is given in Table I. 

 Table I: Typical specifications for catalyst poisoners in polymer grade hydrocarbons (6)			

Mass spectrometry (MS) is hardly used in petrochemical QC laboratories, which is primarily because of its apparent complexity and higher cost-of-ownership. Nonetheless, MS detection has several distinct advantages over classic analog detectors. In full-scan acquisition mode, for example, it allows tracking and identification of unknown components using spectral deconvolution and subsequent library matching. In selective ion monitoring (SIM) mode, on the contrary, MS permits trace and ultratrace quantification of target analytes which is often superior to classic selective detectors. Furthermore, MS permits the use of mass-labeled internal standards that behave identically to their native analogs, which has a positive effect on overall method precision and accuracy. It is no surprise that instrument manufacturers have invested substantially in solutions aimed at reducing overall MS system complexity and total cost of ownership in the past couple of years. Easy tune and calibration functionalities, increased sensitivity and speed, new acquisition modes, and elegant solutions that eliminate downtime, such as vacuum lock technology, have contributed largely in this respect. 

This article gives an overview of the main characteristics and performance of a new gas chromatography–mass spectrometry (GC–MS) analyzer that has been recently developed. The system combines the chromatographic separation power and backflush–Deans heart-cut capabilities of a classic oxygenate analyzer with the orthogonal separation power, sensitivity, selectivity, and overall robustness of the latest generation single-quadrupole mass spectrometers. 

 Standard oxygenate reference mixture from Spectrum at 10 ppm in hexane. For more details with respect to the composition of the test mixture, please consult Table III. Calibration standards were prepared by gradual dilution in hexane at 0.01 ppm, 0.05 ppm, 1 ppm, and 5 ppm.  

 The GC analyzer consisted of a Thermo Trace GC system refurbished by Global Analyser Solutions (GAS). The system was fitted with a gas sampling valve (GSV), a liquid sampling valve (LSV), a vaporizer, a standard split–splitless injector, and an FID system. Inside the GC oven, a universal pressure balanced Deans assembly was installed to carry out heartcut and backflush. Auxiliary pressure for balancing was provided and controlled by a separate Trace GC DCC unit. 

The first-dimension column was a 15 m × 530 µm, 5-µm 

 Restek Rtx-1 column. The second-dimension column was a 10 m × 530 µm, 10-µm 

 Agilent CP-Lowox column. Restrictions were 250-µm i.d. uncoated Siltekdeactivated fused-silica capillary tubing (Restek) cut to the appropriate length. All connections were made using micro Siltite unions (SGE Analytical Science). Other relevant parameters are summarized in Table II. 

 The GC analyzer was hyphenated to a Thermo ISQ single-quadrupole mass spectrometer. The system was applied in both full scan (range: 15–250 amu) and SIM (dwell time: 0.2 s) as full scan–SIM mode. All relevant MS settings are summarized in Table III.  

Table III: Peak identification and typical selective ion monitoring ions			

All data were acquired using Thermo QuanLab Forms software. The MS system was used after running a full electron ionization (EI) tune. System performance was verified using a daily tune check.   

 The capillary column set comprises the true core of any classic catalyst poisoner analyzer. The second-dimension column is particularly important. Ultimately, it is here that separation of the analytes, from each other as well as from the aliphatic matrix in which they reside, occurs. A CP-Lowox column (Agilent) was used for this purpose. This column, which is based on a multilayer porous layer open tubular (PLOT) concept, is very polar and characterized by a high maximum allowable operating temperature with virtually no bleed at temperatures as high as 350 °C (7). In combination with a backflushed apolar-coated capillary column in the first dimension, matrix separations up to C

Unfortunately, the CP-Lowox column is not available in MS-friendly narrow-bore dimensions. To avoid the MS vacuum from protruding the system, it is necessary to incorporate an adequate restriction at the back of the column. A schematic representation of system setup for Lowox/MS applications is depicted in Figure 1. 

Figure 1: Schematic representation of the GCâMS analyzer. BF = backflush; HCs = hydrocarbons.			

True backflush, as well as Deans heart-cut, is achieved by increasing the auxiliary pressure at the column joint just above the first-dimension residual head pressure at a certain moment in time. Debalancing induces full flow reversal, while maintaining a small flow over the Lowox column for chromatography. It is crucial to know the exact pressure at the column joint for this approach to be successful. When the pressure is set too low, standard flow direction will be maintained and the first column is not backflushed. Conversely, when it is set too high, none of the target analytes will be able to reach the second-dimension column and the detector. The easiest way to determine the pressure at the column joint involves setting the head pressure at regular and then reading the residual pressure at the auxiliary digital carrier gas control, which is kept off at this stage. Pressure differences of 5 kPa are sufficient to induce flow reversal. Although less straightforward because of the vacuum conditions, a similar approach is applied in combination with MS. It also permits users to determine the minimal length of the restriction capillary (Figure 1). 

 System suitability was evaluated by direct injection of the 10 ppm oxygenate standard. To compare with a classic analyzer set-up, experiments were performed using both FID and MS for detection. The MS system was applied in both full-scan and SIM modes. A typical chromatogram with the MS in full-scan mode is depicted in Figure 2. Peak identification is referred to in Table III.  

Figure 2: Chromatogram of the oxygenates standard at 10 ppm. The MS system was applied in full-scan mode. Peak identification is referred to in Table III.			

A comparative overview of the results is given in Table IV. For each peak, the signal-to-noise ratio (S/N) was calculated (root mean square [RMS]) in full scan, extracted ion, and SIM mode. These results were subsequently expressed relative to the S/N with FID.  

Table IV: Average sensitivity gains in different MS detection modes. FS = full scan; EIC = extracted ion chromatogram; SIM = selected ion monitoring. %RSD at the 10 ppm level (six analyses).			

The results in Table IV clearly illustrate the significant gains in sensitivity that can be reached when using MS compared to FID. Minimal gain is 3.7 for ethanol. Unsurprisingly, sensitivity gains are particularly significant when the MS was used in SIM mode. Straight full-scan mode proved to be less appropriate for target analysis, which is predominately due to the low molecular weight of the target compounds, and means having to include highly interfering masses such as 

 =18 (water), 28 (nitrogen) and 32 (oxygen) in the scan range. More convenient in this respect is the combined full-scan–SIM mode, which is available on all major instrument brands nowadays. 

Afterward, calibration curves were constructed for each of the oxygenates in the standard mixture from 0.05 ppm to 5 ppm. Correlation coefficients were =0.995. Some typical SIM traces at the 0.10 ppm level are depicted in Figure 3.  

Figure 3: SIM traces at 0.01 ppm: (a) acetaldehyde, (b) ethanol, and (c) propyl ether.			

Method repeatability was determined as well. Results at the 10 ppm level are included in Table IV (six consecutive analyses). 

 Naphtha is a complex mixture of hydrocarbons (C

) in petroleum boiling between 30 °C and 200 °C. Oxygenates are routinely determined in these samples according to reference procedures such as ASTM D7423 (4) as their cracking product causes problems in the downstream separation processes (8). Naphthas are very complex and fully require the chromatographic separation power of the Lowox column. A typical chromatogram of a naphtha sample in SIM mode is depicted in Figure 4. Individual samples were introduced using the LSV of the GC–MS analyzer. The insert shows the methanol trace (ion 29, 0.18 ppm); concentrations of acetaldehyde and TAME were 8.3 and 4.9 ppm, respectively.   

Figure 4: Typical SIM trace of a naphtha feed.			

When idle, the GC oven was kept at 200 °C with the backflush activated. This was necessary to prevent the accumulation of siloxane bleed from the precolumn. 

A GC–MS analyzer is described that substantially expands the workable application range of a classic catalyst contaminants analyzer. The use of MS in full-scan–SIM mode permits identification of unknown contaminants in combination with reliable quantification at trace and ultratrace amounts.  

The authors acknowledge the financial support from the Long Term Structural Methusalem Funding by the Flemish Government – grant number BOF09/01M00409. KVG holds a Postdoctoral Fellowship of the Fund for Scientific Research Flanders and a BOF tenure track position at Ghent University. 

 is a Fulbright alumnus of the Massachusetts Institute of Technology in Cambridge, Massachusetts, and assistant professor in thermochemical reaction engineering in the department of chemical engineering and technical chemistry at Ghent University in Ghent, Belgium.  

is a masters student in civil engineering in the department of chemical engineering and technical chemistry at Ghent University. He is currently finishing his thesis on trace oxygenate analysis using MS techniques. 

 is an analytical support engineer at IS-X. He specializes in instrument design and method development for petrochemical applications.

 is managing expert of IS-X. He specializes in method development and rationalization from prep-to-rep, method validation, and quality assurance.

is chair of the department of chemical engineering and technical chemistry at Ghent University, Belgium. Chemical reaction engineering, catalysis in general, and reaction kinetics are the main topics in his research program.  

(1) L. Resconi, L. Cavallo, A. Fait, and F. Piemontesi, 

(2) Almatis AC Inc., Application for Selective Adsorbents in Polymer Production Processes, Technical Bulletin USA/6040-R00/0504. 

(3) M.A. Graham, "Selected Ethylene Feedstock Impurities: Survey Data," presented at the Ethylene Producers Conference, Houston, Texas, 1993. 

(5) B. Biela, R. Moore, R. Benesch, B. Talbert, and T. Jacksier, Gulf Coast Conference, Galveston, Texas (2003). 

(8) S.P. Pyl, C.M. Schietekat, M.-F. Reyniers, R. Abhari, G.B. Marin, and K.M. Van Geem, 

Article

Ultratrace Quantitative Analysis of Catalyst Poisoners Using a Dedicated GC-MS Analyzer

What do you do when an unexpected peak appears in the chromatogram?

A common liquid chromatography (LC) problem that I get e-mails about from readers of this column relates to unexpected and unwanted peaks that appear in the chromatogram. This month's discussion centers around an e-mail discussion I had with a reader of the electronic version of 

 (available on-line in countries where the paper editions are not available) about a problem they were experiencing. Because this problem relates to a pharmaceutical product and contains proprietary information, I've exercised some obfuscation privileges to hide some of the specifics, but retain the key information. 

The problem relates to the analysis of an antibiotic for impurities. It is a fairly simple method, comprising a phosphate–acetonitrile gradient on a reversed-phase column. The method had been validated and worked quite well to determine a mixture of eight antibiotics and impurities. After successful use in the development laboratory, it was transferred to another laboratory for routine analysis. For identification, I'll refer to these as the R&D laboratory and the production laboratory.  

When the method was transferred to the production laboratory, it appeared to work well, with one exception — a broad peak appeared at a retention time that overlapped the elution of antibiotic 2. In Figure 1, I've shown a section of the baseline including peaks for antibiotics 1 (13.4 min) and 2 (15.6 min) from a chromatogram obtained in the R&D laboratory. This was a typical result for the injection of these two compounds. When the same sample was run in the production laboratory, the results of Figure 2a were observed. The peak for antibiotic 1 appears as normal, but there is a broad peak that all but obscures antibiotic 2.  

An extra peak, that is not related to the sample itself, in a gradient run typically arises from one of three sources:  

late elution from a previous chromatogram

contamination of the mobile phase (ghost peaks).

Many people call all three kinds of peaks ghost peaks, but I like to distinguish between them because, although they may look similar in the chromatogram, their sources are different, and so the process of eliminating them will be different. Let's look briefly at my definitions of each of these peak types. 

 of a peak from a previous chromatogram results when you don't allow enough time for all the peaks to be eluted following sample injection. As a result, the problem peak continues to travel through the column, but shows up in a later chromatogram, instead of the one in which it belongs. 

 from a previous injection is caused by sample that is actually injected, but its injection is unintentional. For example, you make an injection and get a normal run, then you make a blank injection with no analyte present, but the peak appears anyway, usually much smaller than the original. 

 tend to be unique to gradient elution, whereas late elution and carryover are common for isocratic separations, as well. Ghost peaks appear when a contaminant is present in the mobile phase (usually the weak, A-solvent), is concentrated on the column and then is released as the gradient runs. Ghost peaks will appear in sample injections, when a blank is injected, and even when a gradient is run with no injection at all. 

Troubleshooting can be a time-consuming process and unless you have a well-thought-out plan, a lot of time can be wasted and the problem source may be difficult to isolate. I like to make a list of the most probable causes, in this case the potential differences that exist between the two laboratories. Here are the most likely possibilities:  

I'm basically lazy, so I usually apply my "easy vs. powerful" rule of thumb at this point. That is, I'll often do an easy check first, even if it isn't as likely to isolate the problem as a more difficult one. Another practice that I take advantage of is module substitution — replacing a suspect part with a known good one. One of the ways we do this almost instinctively is to make up a new batch of mobile phase or replace the column. Both of these changes were made in the production laboratory, one at a time, which is a good idea if you aren't sure of the problem source. Neither a fresh batch of mobile phase nor a new column solved the problem. 

My contact in the R&D laboratory developed the method on one brand of high performance liquid chromatography (HPLC) equipment, but the production laboratory was using another brand. Part of the question about instrument differences was answered easily when the R&D laboratory transferred their mobile phase and column to the second brand of equipment in the R&D laboratory — it worked just fine. This indicated that there wasn't an inherent instrument design difference that was causing the problem, although other problems, such as contamination could exist. 

Now that the easy things are out of the way, it is time to roll up our sleeves and figure out where the problem lies. Let's look at each of the three peak sources mentioned above and work through them. 

Carryover has something to do with the equipment, usually the autosampler and not the column. A carryover peak is an actual sample peak, not something left on the column from a prior injection. Usually carryover is associated with insufficient flushing of the autosampler between injections, and generates a much smaller peak than normal. For example, after the injection of one sample, an injection of a blank is made and a peak appears at the same retention time as the sample peak, but much smaller. Another blank injection will usually be proportionally smaller than the second injection. Because carryover under normal circumstances is <0.1%, it is very rare to see the same peak appear after two blank injections (0.1% × 0.1% = 0.01%, probably too small to see). Sometimes a sample component can be adsorbed on surfaces of the autosampler tubing or injection valve and will bleed off slowly, resulting in carryover peaks that decline in size only slowly with successive injections, but this is not common for most samples. 

Once again, we can do some mental work, rather than physical, and eliminate this source of the current problem. The peak we observe has an area that has a different retention time, is much larger than our sample peak, and is much broader (note the peak width of the problem peak at 15.2 min compared to the analyte peak at 15.6 min in Figure 2b). If the problem was carryover, the presence of the carryover peak should make the normal analyte peak larger but should not change its area or retention time. So we can eliminate carryover from the probable causes. 

If carryover was the problem, it usually can be corrected by one or both of two fixes. First, use a more thorough autosampler wash routine. Use the strong solvent of the mobile phase (acetonitrile in the present case) as the autosampler wash solvent and increase the number of wash cycles or wash volume between injections. If this does not solve the problem, look for poorly swept passages in the plumbing that might act as tiny reservoirs to trap sample that would then mix with the next injection. Check to be sure that all the tube fittings are seated properly. This is more common when PEEK (polyether ether ketone) fittings are used to make connections. If the system over-pressures, the tubing can slip in these connections, but not enough to come completely loose, leaving a small gap at the tip of the tubing that can trap a small amount of sample. If the injection valve is worn, sometimes it will be less-well flushed and carryover can occur. This is fairly rare, because injector valves are made to cycle 100–500,000 times before a rebuild is necessary. However, if you have >50,000 injections on the valve and have eliminated other potential problem sources, replacement of the injector rotor might be a good idea. 

Late eluted peaks usually are easy to isolate. As a general rule, whether the separation is isocratic or gradient, all the peaks in a given region of the chromatogram should be about the same peak width. If a peak appears that is much wider than its neighbors, the first thing I suspect is that it comes from a previous injection. An example of this is shown for the simulated chromatograms of Figure 3. In Figure 3a, the peak just after 2 min (arrow) is much wider than the neighboring peaks, so it is suspected as a late-eluted peak from a previous injection. In Figure 3b the run time is extended, and now there are two broad peaks, the early one from the previous run and the one at 7 min where it belongs for that injection.  

Late-eluted peaks typically are sample-related — that is, they are sample components that don't have enough time to come off the column. Because the broad peaks were never seen in the R&D laboratory, and the same sample was being analyzed, it is not likely that the problem source is late elution. However, if the problem was isolated to this cause, there are two simple fixes. First, you could extend the run time to allow the peak to be eluted normally. This would work for the example of Figure 3, but would double the run time, which may not be desirable. Another alternative, especially if the problem peak is not of interest, is to flush it off the column with a strong solvent. When a gradient is used, as in the present problem, the column is flushed easily by extending the gradient to 95–100% of the B-solvent (acetonitrile in this case). To speed the process, this can be done as a step. For example, if the normal gradient ran 5–70% B in 20 min, it could be followed by a 70–100% step, a 5-min hold to flush strongly retained material from the column, then a step back to 5% to re-equilibrate for the next injection. 

When gradients are run, any organic material that does not pass through the column under the starting conditions will build up at the head of the column and will be eluted later in the gradient. This is because at the starting conditions (5% acetonitrile in the present case), most organic compounds will have nearly infinite retention, so they just collect on the column as the mobile phase passes through. This is an easy problem to diagnose. Just perform the following sequence of runs. I'll assume for the moment that the normal equilibration between runs is 10 min. 

After you've run this sequence, examine the chromatograms. Ignore the first gradient, because it rarely is equilibrated exactly the same as a normal run. Look at the size of the ghost peaks in the second run (10-min equilibration) and compare this to the same peaks in the third run (30-min equilibration). If the peaks in run 3 grow in the same proportion as the ratio of equilibration times between run 2 and run 3 (threefold in the present example), the source is probably the A-solvent. This is because the longer equilibration allowed three times as much junk to collect before it was eluted from the column. 

When the A-solvent is the problem, each of the components should be checked separately by eliminating them in a stepwise fashion. For example, just use water for A instead of phosphate buffer. If the problem disappears, the phosphate is the probable cause, if not, it is the water. If the chromatogram is better without buffer, try a new bottle of phosphate. Some other possible sources of A-solvent contamination include dirty glassware, the pH meter, and contaminated water. Several of these possible sources are discussed in detail in chapter 17 of reference 1. 

If the above tests seem to confirm the problem source, but careful elimination of mobile-phase components did not solve the problem, another, less common source is left: system contamination. Usually the HPLC system is exposed only to high-purity solvents and reagents, but contamination can occur if you don't practice good laboratory habits. This can be a particular problem when acetate or phosphate buffer are used, because they are good energy sources for microbial growth. If the mobile-phase reservoirs are not replaced with clean ones on a regular basis or if the system is left unattended with buffers standing in the tubing, contamination can occur when bacteria grow in the aqueous phase. Normally, replacement of the reservoir with each new batch of mobile phase and flushing with nonbuffered water or solvent at the end of each day's work are sufficient to keep the system clean. But if you don't regularly practice these preventive measures, any component that contacts the mobile phase can become contaminated, including the reservoir, inlet frits, tubing, degasser, pump, and autosampler. If you've eliminated the obvious causes, an aggressive cleaning may be in order.  

Here's the recommended procedure for aggressive system flushing: 

1. Remove the column, and replace it with a piece of tubing routed to waste. 

2. Make a batch (for example, 0.5 L) of ≈30% phosphoric acid (just dilute concentrated phosphoric acid — 1 part of acid + 2 parts water) and place in a clean reservoir. 

3. Flush all buffers and organic solvents from the system with HPLC-grade water. 

4. Place all inlet lines in the 30% acid reservoir and pump 25–50 mL of acid through each solvent inlet line. Flush the autosampler in both the load and inject positions. Use the same solution and perform multiple injector needle and seal washes. 

5. Remove the acid reservoir and briefly rinse the inlet tube ends and frits with HPLC-grade water. 

6. Fill a fresh reservoir with HPLC-grade water and flush all lines, needle and seal washes, and any other passages exposed to the acid flush. A 25–50 mL water flush should be sufficient, but you can check the pH of the waste line to be sure it is no longer acidic. If the pH is still too low, replace the water and continue to flush. 

This acid-washing procedure was implemented in the production laboratory and a significant improvement was observed, as is seen in Figure 2. In Figure 2b, the relative size of the contaminant peak to antibiotic 2 is greatly diminished (note that for the chromatograms of Figures 1 and 2, the same concentration of standard was used, so the peak height for antibiotic 2 should be the same in all cases; the scale is noted in each chromatogram). It does look like system contamination was at least part of the problem. 

LC Troubleshooting

What do you do when an unexpected peak appears?

Extra Chromatographic Peaks—A Case Study

In this installment we review gas chromatography (GC) instruments and accessories that were newly presented at Pittcon 2012 or were introduced to the marketplace in the preceding year.

The 2012 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (Pittcon) was held March 11–15 in Orlando, Florida, at the Orange County Convention Center. The 63rd annual occurrence of this venerable analytical chemists' symposium was only slightly less attended than the conference's 2010 southern sojourn at the same location, with 15,754 registrants. I'm comparing the attendance to that at the previous Orlando venue rather than Atlanta from 2011 because the attendance always has been somewhat lower when the conference heads south. The amount of exhibitor booth space also declined by a similar margin, driven by many companies' decisions to attend with a smaller footprint, although the total number of exhibiting companies only dropped by about 2%. 

Significantly absent this year were Agilent and PerkinElmer, which are both engaging in postconference seminar tours and on-line product displays. Both companies exhibited at Analytica in Munich, Germany, April 17–20, 2012. Several companies opted to meet key customers at nearby hotel suites rather than hosting an in-exhibition booth. To an aisle wanderer such as myself, the overall impression was one of reduced acreage accompanied by a larger Technology Park area and, although I did not measure them, perhaps slightly wider corridors. Smaller though it may have been, the exhibit floor experience was almost relaxed, with more time to visit each exhibitor of interest, unlike some earlier years where I often had to choose between visiting a booth or attending a session. 

The conference organizers' emphasis on the technical program was evident in the consistent number of poster sessions that were mounted in two large areas of the exhibition floor, as well as in the continuing high count of oral presentations, symposia, and workshops. Short-course attendance was up 16% from last year.  

Among the conferees, 36% listed liquid chromatography (LC) and 34% gas chromatography (GC) among their selected scientific specialties; these were the top two categories at the conference. Spectroscopic specialties accounted for the majority of the remaining mentions; ion chromatography was cited by 11%, and other chromatography techniques were cited by 7%. 

Pittcon 2013 will be held in Philadelphia for the first time, March 17–21, at the Pennsylvania Convention Center. The conference is scheduled to return to Chicago (2014, 2017), New Orleans (2015), and Atlanta (2016), before returning to Orlando in 2018. 

This installment is our annual review of GC instrumentation and accessories shown at this year's Pittcon or introduced during the previous year. For a review of new GC and LC chromatography columns and related accessories, please see Ron Majors's "Column Watch" in the April and May 2012 issues of 

The information presented here is based on manufacturers' replies to questionnaires, as well as on additional information from manufacturers' press releases, websites, and product literature, and not upon my actual use or experience. During Pittcon, I took time to stroll around the convention aisles and see some of the new products firsthand as well as discover a number of items that weren't covered by the questionnaires. Every effort has been made to collect accurate information, but because of the preliminary nature of some of the material, 

 cannot be responsible for errors or omissions. This column cannot be considered a complete record of all new GC products introduced this year at Pittcon or elsewhere because not all manufacturers chose to respond to the questionnaire, nor is all of the submitted information necessarily included here because of the limited available space and the editors' judgment as to its suitability.  

Another new crop of GC instruments appeared in 2012, which demonstrates once again the ongoing resilience of the technique and also emphasizes a continuing shift toward mass-selective detection since what used to be top-end capabilities have become more affordable. Nearly half of the newly introduced instruments are tightly associated with a mass spectrometric (MS) detector. Most were more about the MS detector than the GC system itself. See Table II for the details of each new product. 

Shimazdu's GCMS-QP2010 SE system is an upgraded version of the company's QP2010 S system that features high-performance ion optics and a quadrupole mass filter along with lab-friendly energy- and time-saving features. While not seen at Pittcon, PerkinElmer earlier this year introduced the Clarus SQ 8 GC/Mass Spectrometer system with high sensitivity, tool-free source maintenance, and a rapid scanning speed. Bruker Daltonics highlighted its high-end Scion TQ GC–MS-MS system with 180° optics that enables multiple ion-selective scan modes for resolution of complex samples and matrices. This triple-quadrupole GC–MS system, introduced in July 2011, won the 2012 Pittcon Editors' Silver Award. All of these new GC–MS systems operate in tandem with the companies' respective existing gas chromatograph offerings. 

From Torion, the Tridion-9 GC-TMS system is a gas chromatograph–toroidal mass spectrometer system that is aimed at the rapid screening of chemicals such as environmental volatile organic compounds (VOCs) and semivolatile organic compounds (SVOCs), explosives, chemical threats, and hazardous chemicals. The unit is billed as a "Person-Portable" GC–MS system that can easily be carried to remote sites. It uses a conventional 100-µm i.d. capillary column bundled with a low-thermal-mass resistive heater and helium carrier gas. Sample injection is accomplished via the company's Custodion solid-phase microextraction (SPME) syringe. 

Thermo Fisher Scientific came to Pittcon with the 1300-Series GC system, a completely new frame. With a unique system of "instant-connect" modular interchangeable inlet and detector options, the 1300-Series system allows end users to quickly exchange or replace either or both inlets or detectors as pluggable modules. Each module includes pneumatic and electronic connections that are engaged by putting them in place and tightening a couple of screws. 

PAC introduced the AC Analytical Controls CNS SIMDIS for Crude Oil analyzer. The system simultaneously determines the boiling range distribution of carbon, nitrogen, and sulfur in crude oils and final products, using simulated distillation and a combination of flame ionization detection (FID) and chemiluminescence detection. 

A unique portable GC instrument was on display at the 2012 Pittcon. The Frog-4000 system from Defiant Technologies is a miniaturized GC system with photoionization detection (PID). Based on micromachined preconcentrator and GC designs, the Frog-4000 system targets field analysis of VOCs in water and soil.  

This year saw strong growth in new automated headspace GC (HSGC) samplers. Together with Agilent's 7697A headspace sampler that was introduced at Pittcon 2011, this burgeoning of HSGC is indicative of the ongoing demand for volatiles analysis in solid, semisolid, and liquid matrices such as for pharmaceutical and food or flavor products. Table III lists the salient details.  

A review of gas chromatography instruments and accessories presented at Pittcon 2012

LCGC North America-05-01-2012

Bioanalytical Tools for the Characterization of Biologics and Biosimilars

Column Characterization Databases

New Chromatography Columns and Accessories at Pittcon 2012, Part II

Ultratrace Quantitative Analysis of Catalyst Poisoners Using a Dedicated GC-MS Analyzer

Extra Chromatographic Peaks—A Case Study

New Gas Chromatography Products at Pittcon 2012

Vol 30 No 5 LCGC North America May 2012 Regular Issue PDF