This installment focuses on advanced analytical separations that incorporate nanomaterials for improved chemical selection. These new nanomaterials are based on bioinspired and polymeric self-assembled aggregates. Capillary electrophoresis and chromatographic methods were harnessed for the separation of a wide variety of samples. Compelling applications include rapid separations of 100 DNA fragments and separations of enantiomers, aptamers, small molecules, and glycans.  The implementation, performance, and fundamental properties of these new media are discussed.  

The Pittsburgh Conference of Analytical Chemistry and Applied Spectroscopy (Pittcon), was held March 12–18, 2011, at the World Congress Center in Atlanta, Georgia. The Pittcon 2011 technical program boasted a number of exciting advances in analytical chemistry, including a number of leading edge technologies in the area of separation science. A session sponsored by the American Chemical Society's (ACS) Subdivision of Chromatography and Separations Chemistry on self-assembled nanomaterials for enhanced chemical separations was organized by Professor Lisa Holland of the Department of Chemistry, West Virginia University (Morgantown, West Virginia).  

Presentations in this Pittcon session outlined enhanced separations and diverse applications with techniques based on new materials for thin-layer chromatography (TLC), solid-phase microextraction (SPME), and capillary electrophoresis (CE). Some separation media discussed in this session emanated from fabrication techniques such as photolithography and chemical vapor deposition. A new "spin" on fabrication was nanofiber electrospinning. Self-assembly of biomolecules inspired by natural systems was a prominent theme. Generally, these biological selectors were effective for the separation of biopolymers such as DNA, aptamers, and glycans, as well as biologically relevant small molecules. Classical self-assembled surfactant micelles were adapted to improve electrically driven separations.  

The purpose of this installment of "Column Watch" is to summarize some of the key techniques presented in this session and discuss future directions that new self-assembled nanomaterials may take in separation science. 

Background in Self-Assembled Nanomaterials for Separation Science

Traditional separation media such as chromatographic packing material or open-tubular chromatography columns are fabricated through the covalent modification of a polymeric or inorganic (silica, for example) surface. With new advances in material science, newer strategies arise that result in sophisticated architecture for chemical separations based on molecular self-assembly. These processes result from spontaneous molecular interactions to control the size, shape, or surface characteristics of the assembly, often resulting in what is a highly ordered and unique structure. A benefit of this approach is that reduced chemical modification results in a greatly simplified fabrication protocol of what is a complex final product. Assembled structures may be made from components that are inorganic or organic (or both) or biological in nature. Frequently, such materials originate from or are inspired by biological systems, such as cellular membranes. The resulting supramolecular assembly can occur through a variety of mechanisms such as ionic, hydrophobic, or hydrophilic interactions, as well as adsorption. Although individual interactions may be weak, the summative force from these interactions stabilizes the entire assembly. These molecular assemblies may possess nanoscale features as well as unique properties that macroscale materials often lack.  

Nanofabrication: The Patterned Nanotube Forest or the Imprinted Nanofiber?  

David Jensen and colleagues from the Department of Chemistry and Biochemistry at Brigham Young University (BYU, Provo, Utah) reported ongoing work in carbon nanotube–templated porous materials. Prototype devices have been constructed with a variety of nanofabrication methods. The methods, as discussed at the Pittcon session (1) and outlined in the literature (2), provide control of both the 3-D microscale shape as well as the porosity of what might be described as a patterned carbon nanotube forest. These materials can be used as high-tech silica-based TLC plates. In the session, Jensen presented several custom-fabricated normal-phase TLC plates that incorporated a herringbone pattern (Figure 1). This pattern facilitated higher separation efficiencies and shorter run times as compared to state-of-the-art commercially available TLC plates. This work was done in the laboratory of Matthew Linford at BYU and was also directed by Robert Davis in the BYU Physics department. It was sponsored by US Synthetic Corp. (Orem, Utah), which intends to commercialize the work. 

 Figure 1: Depiction of thin-layer chromatography based on the herringbone pattern shown in the SEM image in the inset that is achievable with silicon-carbon core-shell nanotubes. 

A twist on previously reported ultrathin-layer chromatography (3,4) based on electrospun carbon nanofibers was introduced by Susan Olesik and coworker Joseph Zewe of Ohio State University (Columbus, Ohio). As depicted in Figure 2, nanofibrous stationary phases are produced through electrospinning different polymer solutions from a syringe attached to a high-voltage power supply onto a grounded substrate surface. In TLC, the resulting stationary phase provides superior separation efficiencies to commercial counterparts. Electrospinning also can be used to fabricate unique self-assembled and self-polymerizing molecular imprinted fibers (5). The work presented at Pittcon 2011 (6) centered on a solid, yet ultrathin mat of carbonaceous material imprinted with the chemical warfare agent surrogate dibutyl butyl phosphonate. With simple electro-spinning and pyrolysis, the imprinted material was incorporated into an SPME material and used to extract dibutyl butyl phosphonate from both simple and complex matrices. In both cases, with and without interferences, imprinted SPME media demonstrated enhanced selectivity for the template molecule, extracting more warfare simulant than nonimprinted SPME fibers. Molecularly imprinted carbon is both highly chemically and thermally stable, which are also advantages over traditional molecularly imprinted polymers. 

Figure 2: Schematic of the straightforward process for electrospinning nanofibers adapted from reference 2. 

Bioinspired Self-assembled Materials: If Nature Will Not Be Replicated in the Laboratory, Can It Be Plagiarized? 

Separation materials derived or modeled from those discovered in biological sources offer a wide variety of functionality as separation material. Guanosine gels, for example, reversibly self-assemble as a function of temperature, compound concentration, solution composition, and pH (7). These materials have previously been harnessed polymorphic to separate single-stranded DNA in capillary gel electrokinetic chromatography based on the DNA sequence. In a new application reported in the session by Yingying Dong of Rensselaer Polytechnic Institute (Troy, New York) (8), the unique morphology of this medium was harnessed for the separation of chiral enantiomers (9). The self-assembled materials form columnar "G-wires" stabilized by π-π interactions, leading to helical, asymmetric structures. When integrated in CE, the authors were able to separate enantiomers of molecular weight <300 Da. The material is inexpensive and simple to prepare, and the method provides reproducible migration times. Baseline resolution was achieved within 10 min and 16 min for enantiomers of tryptophan and BNPA, respectively (see Figure 3). Interestingly, not all enantiomers could be separated, leading to structural studies of the interactions between the G-gels and chiral compounds that will provide a basis for modifications of the G-gels to achieve chiral separations for a wider range of compounds. 

Figure 3: Capillary electrophoresis separations of tryptophan enantiomers (top) and BNPA enantiomers (bottom) using guanosine-5'-monophosphate run buffer (0.05 M GMP and 0.05 M KCl in 25 mM potassium phosphate buffer, pH 7.0). 

Another bioinspired self-assembled material used for CE separations was presented by the Holland laboratory at West Virginia University. The phospholipids dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC) can self-assemble into different morphologies and offer a number of advantages including a thermally responsive viscosity. At room temperature, preparations of these phospholipids have a viscosity similar to that of water. However, at temperatures a few degrees higher preparations exhibit a significantly increased viscosity (10). An important consequence of this thermal transition is that the material can be easily loaded into the capillary at temperatures below 24 °C, facilitating a gel-like separation in CE with a greatly simplified column preparation method. Following separation of the analytes at the elevated temperature, the capillary temperature can be dropped, and the phospholipid can be expelled and replaced as needed. One application is the separation of single- and double-stranded DNA. Lisa Holland and coworkers (11) demonstrated the utility of phospholipid additives to efficiently separate solutions of double-stranded DNA that vary in length by only a few base pairs. As shown in Figure 4, the method was also used to identify conformational changes of single-stranded DNA aptamers and to probe binding of an aptamer to its selected target. Another benefit of the high viscosity of this thermally responsive phospholipid media is that other selection agents can be incorporated into the material and immobilized in the separation capillary without any covalent methods of binding. In addition to conformational change, the material has been loaded with aptamer target to retard the aptamer migration through the media, thus demonstrating molecular affinity. 

Figure 4: Example of the use of a phospholipid capillary separation integrated with thrombin to capture thrombin-binding aptamer. The two traces show the separation of a single-stranded DNA aptamer that is a 15-mer specific for thrombin separated in the presence (lower trace) and absence (upper trace) of the thrombin plug. 

This medium also proved ideal to separate glycans (12,13), which are branched oligosaccharides relevant to numerous biological studies and applications. When glycans are derivatized with the fluorescent label 8-aminopyrene-1,3,6-trisulfonic acid (APTS), they may be detected using laser-induced fluorescence at low limits of detection and at a labeling stoichiometry of 1:1, facilitating quantification. Additionally, the APTS label imparts negative charge to the glycan molecule. When the glycan is neutrally charged, for example following desialylation, the derivatization enables glycan separation based on hydrodynamic volume. The added viscosity provided by phospholipid media enhances the separation of these glycans, even resolving glycans with the same monomer composition and sequence, but differing only in a single terminal linkage. When integrated in a 25-µm i.d. capillary, theoretical plate counts in excess of 600,000 are obtained. Stephanie Archer-Hartmann (14) and colleagues from West Virginia University presented an application of this separation method by assessing complex mixtures of glycans derived from glycoprotein standards and from biological samples through the incorporation of proteins such as lectins or enzymes directly into the capillary. Examples included the lectin concanavalin A, which binds strongly to high mannose glycans, as well as exoglycosidases that remove terminal mannose and galactose from the glycan. All of these separations were accomplished without the need for covalent modification of either the capillary or the protein. Archer-Hartmann demonstrated the use of this method to quickly survey glycoproteins derived from the MCF7 immortalized human breast cancer tumor cell line with the intention of efficiently separating, but also discerning, structural characteristics of the derived glycans. 

A Return to the Micelle: Unique Separation Mechanism with Self-Assembled Surfactants 

For more than 25 years, the role of micelles as a vehicle to support partitioning in electrokinetic separations has been established (15), and yet, the role of micelles in electrophoresis has expanded again. James W. Schneider of Carnegie Mellon University (Pittsburgh, Pennsylvania) reported on a novel, gel-free method to electrophoretically separate DNA oligomers using nonionic surfactant micelles (16). The oligos are tagged by hybridization of an end-alkylated peptide-nucleic acid (PNA) surfactant to their 5' end (17) or to both 5' and 3' ends (18). These peptide nucleic acid–tagged oligos are then separated in CE using a running buffer containing approximately 3 vol% of nonionic surfactant micelles. During the run, micelles bind the peptide nucleic acid tags to retard the electrophoretic mobility of the oligomers in a length-dependent fashion. Because gels are not used, the run times are 10–100 times faster than capillary gel electrophoresis and are compatible with commercially available CE instrumentation. The key to success of the method is a rapid fluctuation of micelle size during the run, so that highly uniform drags are experienced by all oligos in the sample. A model was presented predicting run times for various applications, including DNA sequencing, identification of STR loci, and mRNA profiling.  

Finally, in covering the development of self-assembled materials to improve chemical separations, Charles Lucy and colleague Bob Stanley of the University of Alberta (Edmonton, AB, Canada) addressed the disassembly of self-assembled media (19). Micellar electrokinetic chromatography (MEKC) is a powerful separation technique, but surfactants strongly suppress ionization in electrospray mass spectrometry (MS) making MEKC–MS challenging. The Canadian workers explored the model acid-labile surfactant, sodium 4-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl) methoxy]-1-propane sulfonate (ALS), as a media for micelle electrokinetic capillary chromatography separations (Figure 5). The optimal conditions for bulk synthesis and separation were characterized. Efficiencies of greater than 100,000 plates and migration time reproducibilities comparable to sodium dodecyl sulfate were achieved in ALS-MEKC. After separation, the ALS can be hydrolyzed off-line (

 = 48 min) to nonsurface active products compatible with electrospray ionization. 

Figure 5: Acid hydrolysis of the surfactant ALS. 

The Future of Self-Assembly Nanomaterials: Could Be in Discovery of Nanomaterials That Provide an Added Function to the Analyte Separation 

Additional talks during this session at Pittcon 2011 included presentations on novel nanomaterials to enhance methods of detection and optical methods of analysis. At Louisiana State University (Baton Rouge, Louisiana), such innovative nanomaterials could ultimately be the secret ingredient hidden in the group of uniform materials based on organic salts (GUMBOS), according to Sergio L. de Rooy (20). These nanomaterials with thiacarbocyanine compounds originate from a group of uniform materials based on organic salts that are prepared via an ion-exchange reaction. For these nanomaterials distinct morphologies exhibited spectral properties based on Foster resonance energy transfer as well as organic dye aggregation. Jiao Chen and colleagues (21) of the University of North Dakota (Grand Forks, North Dakota) reported the production of metallic nanostructures to enhance fluorescence intensity with applications in a number of different bioanalytical studies. From Korea University (Seoul, South Korea), Man Bock Gu and colleagues (22) conveyed the role of the affinity ratio in colorimetric nanoparticle-based aptasensors. The utility of the approach was demonstrated by the selective detection of five different small molecule–based targets. Nancy Xu of Old Dominion University (Norfolk, Virginia), gave a presentation on nanoparticles as "nano rulers" (23) and as probes of size-dependent transport kinetics of receptors in cells (24). Can new materials hold potential to sense and separate? Perhaps the answer will be revealed at Pittcon 2012.  

(1) D. Jensen, A. Dadson, M. Linford, M. Vail, R. Vanfleet, R. Wyman, R. Davis, and S. Kanyal, Pittcon 2011, Paper 115-7, March 13, 2011. 

(2) J. Song, D.S. Jensen, D.N. Hutchison, B. Turner, T. Wood, A. Dadson, M.A. Vail, M.R. Linford, R.R. Vanfleet, and R.C. Davis, 

(5) J.W. Zewe, J.K. Steach, and S.V. Olesik, 

(6) S.V. Olesik and J. Zewe, Pittcon 2011, Paper 815-1, March 15, 2011. 

(7) Y. Yu, D. Nakamura, K. DeBoyace, A.W. Neisius, and L.B. McGown, 

(8) Y. Dong and L. McGown, Pittcon 2011, Paper 815-3, March 15, 2011. 

(9) W.S. Case, K.D. Glinert, S. LaBarge, and L.B. McGown, 

(11) L.A. Holland, B. Durney, and S. Archer-Hartmann, Pittcon 2011, Paper 115-6, March 13, 2011. 

(12) R.J. Luo, S.A. Archer-Hartmann, and L.A. Holland, 

(13) S.A. Archer-Hartmann, L.M. Sargent, D.T. Lowry, and L.A. Holland, 

(14) S.A. Archer-Hartmann, L. Holland, and X. Wu, Pittcon 2011, Paper 115-1, March 13, 2011, 

(15) S. Terabe, K. Otsuka, K. Ichikawa, A. Tsuchiya, and T. Ando, 

(16) J. W. Schneider, A. Holmen, M. Fahrenkopf, and S. Istivan, Pittcon 2011, Paper 815-2, March 15, 2011. 

(17) S.T. Grosser, J.M. Savard, and J.W. Schneider, 

(18) J.M. Savard, S.T. Grosser, and J.W. Schneider, 

(19) C.A. Lucy and B. Stanley, Pittcon 2011, Paper 115-8, March 13, 2011. 

(20) S.L. de Rooy, B. El-Zahab, I. Warner, M. Li, and S. Das, Pittcon 2011, paper 115-3, March 13, 2011. 

(21) J. Chen, N. Fahrudding, W. Thompson, and Y. Jin, Pittcon 2011, Paper 115-2, March 13, 2011. 

(22) M.B. Gu, J.H. Kim, S.J. Lee, and Y.S. Kim, Pittcon 2011, Paper 115-4, March 13, 2011. 

(23) N. Xu, P. Nallathamby, and T. Huang, Pittcon 201, Paper 815-4, March 15, 2011. 

(24) N. Xu, K. Lee, and P. Nallathamby, Pittcon 2011, Paper 115-5, March 13, 2011. 

"Column Watch" Editor Ronald E. Majors is Senior Scientist, Columns and Supplies Division, Agilent Technologies, Wilmington, Delaware, and is a member of LCGC's editorial advisory board. Direct correspondence about this column via e-mail to 

Articles

Column Watch

Nanomaterials offer exciting possibilities for improving separations with techniques such as TLC, SPME, and CE.

Self-Assembled Nanomaterials for Enhanced Chemical Separations

Ghost peaks can be difficult to eliminate when minor peaks are important in an LC separation. This case study shows how to isolate the source of such peaks.

Ghost peaks can be referred to as artifact (erroneous) peaks, system peaks, pseudo peaks, vacancy peaks, eigenpeaks, induced peaks, or spurious peaks (1). These are often observed unexpectedly in a chromatogram and can pose a challenge to analytical scientists. These peaks may arise from unknown impurities or artifacts within the liquid chromatography (LC) system (such as contaminants from a dirty injector needle, an air bubble in the pump, or a trapped contaminant in the guard column), from the mobile phase, from an extractable contaminant, from autosampler vials and caps, or from a contaminant carried over from a previous injection. 

The occurrence of ghost peaks is seen more frequently in the gradient mode. The presence of a ghost peak in a sample chromatogram, especially at the elution time of a known impurity, may trigger an out-of-trend (OOT) or out-of-specification (OOS) investigation. An OOT or OOS investigation will lead to efforts to identify an assignable cause, assess the impact of the result, and propose further corrective or preventive action, if required. Because the contamination or artifact causing the ghost peak may come from many sources or may be transient in nature, the investigation can be time-consuming. In certain cases, the cause of the ghost peak may remain unresolved. 

Furthermore, in some company standard operating procedures (SOPs) in a good manufacturing practice (GMP) environment, if the cause of a random ghost peak cannot be identified and the peak is ruled out as an impurity unrelated to the sample, the data may need to be reported "as is." In some situations a blank subtraction can be justified when reporting the data with ghost peak problems; however, this is usually not preferred in routine quality control (QC) laboratories. The biggest problem with significant ghost peaks is that they make automatic integration of chromatograms difficult. Hence, when manual integration or blank subtraction is used, ghost peaks can cause inconsistent and inaccurate impurity results. The problem eventually intensifies if the inconsistent impurity result has been used for trending purposes in stability studies.  

Many publications (including past installments of this column) have cited ghost peak problems and perspectives (2). Here, we outline a step-by-step approach we have used to investigate the source of ghost peaks in a gradient LC method and share our empirical perspectives as quick approaches for solving ghost peak problems in LC. As a case study, we'll offer our experience with a reversed-phase LC method that was used to analyze a nonpolar, small-molecule active pharmaceutical ingredient (API) in an early development stage.  

Whenever gradient methods are used, it is a good idea to run a blank gradient, in which no injection is made or only the sample diluent is injected, to check for any ghost peaks that might be present. In this study, acetonitrile was used as the sample diluent. An overlay chromatogram is shown in Figure 1 that compares injections of an acetonitrile blank with multiple ghost peaks and an injection of the API sample containing related substances. The analyses were performed on a Waters LC Alliance 2695 and Waters photodiode-array detector model 2996 (Waters Corporation, Milford, Massachusetts) with a 250 mm x 4.6 mm, 5-µm Luna C8 column (Phenomenex, Torrance, California) operated at 25 °C and 1 mL/min, with 264-nm UV detection. A linear gradient was run from 95% 50 mM ammonium acetate buffer (pH 4.75) (mobile phase A) to 95% acetonitrile (mobile phase B) in 30 min, with a final 5-min hold at 95% acetonitrile. 

 Figure 1: Overlay of chromatograms with ghost peaks (circled) from a blank (lower chromatogram) that interfere with an impurity sample analysis (upper chromatogram).			

The overlay chromatogram of Figure 1 clearly shows that some of the ghost peaks in the blank (lower chromatogram) were eluted at the same time as some of the related substances (upper chromatogram); these will interfere with the determination of the related substances.  

As shown in Figure 2, multiple runs on different days indicated that these ghost peaks showed up inconsistently in various acetonitrile blank injections. This random appearance of ghost peaks made related substance peak identification difficult. Furthermore they led to an elevated baseline (or a bump) that made peak integration more difficult. 

Figure 2: Overlay of chromatograms of acetonitrile blanks injected on different days, showing the inconsistent nature of the number and size of ghost peaks.			

To isolate the source of the ghost peaks, we eliminated one potential source at a time until we found the root cause. 

Systematic experiments were performed to rule out mechanical sources within the LC system by cleaning the LC system components that may potentially cause ghost peaks. Generally, these experiments are described in the LC system manufacturer's troubleshooting guidelines (3). In our investigation, we took the following steps: 

1. Clean the LC system by flushing with 25:25:25:25 water–isopropanol–acetonitrile–methanol at flow-rate of 1 mL/min for 1–2 h (without the column attached). This procedure helps flush away any containments over a wide polarity range. 

2. Change to a new in-line filter or guard column (or both), if applicable. This will ensure that the LC system is free from any potential contaminants deposited on the in-line filter or guard column. 

3. Clean and purge the injector needle (to rule out a dirty injector or air peak). Typically, a needle wash solution mixture of equal volumes of water and organic solvent (acetonitrile, methanol, or isopropanol) was used to clean the needle or needle seat manually or automatically, depending on the LC system used. The cleanliness of the injector needle can be checked by running a blank gradient run (with or without an actual injection). For Waters's LC systems, a "condition column" function performs a no-injection gradient. 

4. Clean and wash the UV detector cell. This can be done by following the detector manual instructions. Be careful not to exceed the UV cell's pressure limit. 

5. Check the intensity of the UV lamp (to rule out a failing lamp). 

After ensuring that the LC system modules were clean, blank acetonitrile was injected into the cleaned system using our gradient method. Ghost peaks were still observed with freshly prepared mobile phases, indicating that the causes of the ghost peaks were not a result of mechanical sources from the LC system.  

 Contamination from Glassware and Filtration of Reagents 

To rule out any contamination from glassware, we tested several mobile phase bottles by rinsing them and by testing the mobile phase either with or without sonication. Initially, a blank injection was performed by using the mobile phase contained in a bottle (without sonication). Subsequently, the same bottle containing the mobile phase was placed in a water-bath sonicator. After 30 min of sonication at ambient temperature, a second blank injection using the sonicated mobile phase was performed, and the results from the two runs were compared. 

From the sonication we expected to extract any additional amounts of the source contaminant from dirty glassware (the bottle) into the mobile phases. This study was repeated several times with different preparations of mobile phases. No significant changes were observed in sonicated and unsonicated mobile phases, which indicated that the mobile phase glassware was not the source of ghost peaks. 

A blank injection was also performed using a freshly prepared mobile phase A (ammonium acetate buffer) that was passed through a 0.45-µm nylon filter to evaluate whether filtration introduced any contaminants. As a control, the acetonitrile blank was injected using unfiltered mobile phase A. Filtration of buffered mobile phases reduced the areas of ghost peaks slightly, and that suggested that mobile phase reagents could be the source of ghost peaks.  

Contamination from Mobile Phase A Reagents and Solvents

The effect of high performance liquid chromatography (HPLC)-grade water on ghost peaks was evaluated using water purified by a Milli-Q system (Millipore Corp., Billerica, Massachusetts) and commercially available HPLC-grade bottled water in preparing mobile phase A (ammonium acetate buffer). We did not see any significant differences between the two blank injections when comparing sources of water in the mobile phase. This led us to suspect that the ghost peaks could be arising from the organic reagents or additives in the mobile phases.  

First, we evaluated the glacial acetic acid that we used in preparing ammonium acetate buffer. Five HPLC-grade acetic acid brands were evaluated while keeping the ammonium hydroxide and acetonitrile (mobile phase B) brands unchanged. Figure 3 demonstrates that some of the ghost peaks (at 23-, 25-, and 30-min retention times) resulted from impurities in a specific acetic acid brand (brand 1) used to prepare the mobile phase A. Another ghost peak at 15 min showed up primarily in brand 2 and to a minor extent in brand 3. 

Figure 3: Comparison of blank chromatograms comparing acetic acid brands when all factors except the source of acetic acid were held constant.			

The next step was to use the glacial acetic acid (brand 5; the cleanest) that gave no ghost peaks, and to compare sources of the next reagent used in preparing mobile phase A. Acetonitrile blanks were injected while changing the brands of ammonium hydroxide. Interestingly, another ghost peak at a retention time of about 11 min resulted from one source of ammonium hydroxide, as shown in Figure 4. At this point, we had successfully pinpointed the source of five ghost peaks (at about 11, 15, 23, 25, and 30 min) that came from the particular brands of acetic acid and ammonium hydroxide used to prepare the mobile phase A buffer when the problem originally appeared. This can be seen by comparing the chromatograms generated with acetic acid brand 1 (Figure 3) and ammonium hydroxide brand A (Figure 4) with the blank runs of Figures 1 and 2. 

Figure 4: Blank chromatograms from two sources of ammonium hydroxide; all other factors were held constant.			

Contaminant from Mobile Phase B (Organic Solvent)

By a process of elimination, the remaining unidentified ghost peak that was eluted at 24 min (Figure 1) was suspected to come from mobile phase B (acetonitrile). To prove that this peak was not coming from mobile phase A, a blank was injected using Milli-Q water instead of ammonium acetate buffer (as mobile phase A) and acetonitrile (as mobile phase B), as shown in Figure 5. Then two HPLCgrade brands (X and Y) of acetonitrile were compared, as shown in Figure 6. Based on these overlay chromatograms, we are certain that acetonitrile brand X (our original source) contributed to the ghost peak that was eluted at 24 min. Lastly, by choosing the selected ammonium hydroxide, acetic acid, and acetonitrile brands with no ghost peak contributions, a blank sample was injected to produce a clean baseline (Figure 7). The tiny peak at 30 min did not interfere with impurities that had been identified in our samples, so this was acceptable. 

Figure 5: The effect of elimination of ammonium acetate in mobile phase A.			

We successfully isolated the source of all major ghost peaks in our LC method by using a systematic elimination approach. In our case, the ghost peaks were coming from both mobile phases. After ensuring that all the mechanical aspects of the LC system are functioning properly, it is appropriate to shift the investigation directly to the mobile phases. We recommend evaluating different brands of HPLC-grade reagents, because some can contain impurities that can cause ghost peaks in specific LC methods. 

Figure 6: Comparison of blank chromatograms obtained from two different sources of acetonitrile; all other factors were held constant.			

As preventive action, we suggest specifying the vendors and part numbers of solvents and reagents used in preparing mobile phases in the analytical procedure. This information is very useful during method development and especially in method transfer so that ghost peak problems can be prevented. However, if the method specifies an "equivalent" brand of reagent, it is not always necessary that the replacement reagent be of optimal grade or expensive. In such a situation, one needs to evaluate the most suitable reagents for the assay because each contaminant arising from reagent impurities may have a different UV absorption and may only be a problem under specific conditions and not others. 

Figure 7: Successful elimination of interferences (top chromatogram) after selecting uncontaminated reagents (bottom chromatogram).			

Sometimes the cause of ghost peaks remains unresolved and the peaks are impossible to eliminate. In such cases, three possibilities exist:  

1. For a ghost peak that is consistently present and is eluted at a specific retention time, but does not overlap with any peak of interest, the contribution of the ghost peak's area (obtained from blank injections) can be ignored. 

2. For a ghost peak that is inconsistently present, but is eluted at a specific retention time and does not overlap with any peak of interest, the identified contaminant can be used as a retention time marker in system suitability testing and excluded from reporting as an impurity. This approach may only be used after identifying the source of the contaminant. For example, a common contaminant may come from widely used plasticizers that may easily (but inconsistently) leach and extract out from a variety of products into the blanks and sample solutions. 

3. For a ghost peak that is consistently present and is coeluted with a peak of interest, the ghost peak's average area response in blank injections bracketing the sample injections can be subtracted from the sample chromatograms to obtain the net peak area, but only if the overlapping ghost peak has consistent area response throughout the entire LC analysis batch. This approach can be used provided that the ghost peak is present in all blank injections and is not sample-related. It should be noted that such background subtraction is much less desirable than eliminating the interfering ghost peak by isolating its source or moving it out of the way in the chromatogram.  

In any case, the outcome of the ghost peak investigation should be well documented and reflected in the test procedure if necessary.  

 (Humana Press, Clifton, New Jersey, 1989). 

, American Laboratory and Waters Corporation, 720000181EN, 08/02. 

 is a formulation development scientist at SuperGen in Pleasanton, California, working with early LC method development, preclinical and clinical preformulation, and formulation development. 

 is an analytical scientist at SuperGen working with R&D formulation and analytical personnel on R&D and clinical stability studies. 

 is the director of analytical development at SuperGen and is responsible for the release and stability testing of drug substances and drug products. 

 is the vice president of manufacturing and preclinical development at SuperGen. 

LC Troubleshooting

Here is a step-by-step approach to investigate the source of ghost peaks in a gradient LC method.

Ghost Peak Investigation in a Reversed-Phase Gradient LC System

The Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (Pittcon) is a showcase for the latest advances in laboratory instrumentation and technology, attracting all of the major high performance liquid chromatography (HPLC) vendors and a total of more than 1000 exhibiting companies from around the world. Pittcon is a great place for vendors to introduce, and for scientists to see, touch, evaluate, compare, and hear all about the latest in HPLC technology. This installment of "Innovations in HPLC" highlights some of the new HPLC and related technology introduced at this year's conference. 

Because Pittcon attracts upwards of 20,000 attendees from industry, academia, and government from more than 90 countries worldwide, the conference provides a great opportunity for vendors to expose new high performance liquid chromatography (HPLC) products to both new and existing customers. With this fact in mind, many HPLC vendors have for years coordinated their new product development cycle around Pittcon, introducing new technology at the conference for the first time. Although the proliferation of smaller, specialized conferences has altered this approach somewhat in recent years (for example, many new mass spectrometry [MS] product introductions are now made at the American Society for Mass Spectrometry annual conference), Pittcon still remains a must-attend meeting to see the latest and greatest in HPLC and related technology. For the attendees, the conference provides a venue to evaluate the latest instrumentation, compare vendors, participate in product demonstrations, and speak with technical staff to resolve problems or investigate potential applications. 

At this year's Pittcon, several vendors introduced new systems and components, as well as product line extensions. In this installment of "Innovations in HPLC," I'll review some of the new HPLC instrument technology shown at the conference; Table I lists the introductions reviewed in this column. (New column and sample preparation technology is reviewed in 

 by Ron Majors in his "Column Watch" column.)  

Table I: Summary of systems and components reviewed in this article			

Sometimes referred to as biotech drugs, biopharmaceuticals, or biotherapeutics, biologicals are protein- or peptide-based therapeutics (also included are nucleic acids, whole cells, monoclonal antibodies, viral particles, and vaccines) derived from either genetically altered bacteria or fungi or from blood and blood plasma products. Recombinant proteins and monoclonal antibodies (mAbs) are examples of two of the most prevalent biologics; follow-on biologics, or biogenerics, is another area garnering increased attention. Many pharmaceutical and biotechnology companies are continuing to look for new ways to improve the formulation and delivery of biologic drugs. Because this area is the fastest growing market segment in the pharmaceutical industry, several HPLC instrument vendors have begun to focus on this growing market segment, as evidenced by Pittcon 2011 product introductions. Many Pittcon 2011 instrument introductions for biologics analysis were extensions of existing instrument platforms (ultrahigh-pressure liquid chromatography [UHPLC] remains a growing technology platform for many vendors), while some were based on new instrument technology.  

Waters (Milford, Massachusetts) introduced three offerings in this area: the Acquity UPLC H-Class Bio system, the NanoAcquity UPLC system with 2D Technology, and the NanoAcquity UPLC system with HDX Technology. Waters showcased its next-generation UHPLC instrument called the Acquity UPLC H-Class system at Pittcon in 2010. The Acquity UPLC H-Class Bio System advances macromolecule analysis through multiple-mode chromatographic capability, automated quaternary solvent blending, and the use of corrosion-resistant inert materials. Designed for the analysis of proteins, peptides, nucleic acids, and glycans, it has the flexibility to perform four chromatographic modes on a single system: reversed-phase chromatography, ion-exchange chromatography, size-exclusion chromatography (SEC), and hydrophilic interaction chromatography (HILIC). A key feature of the system is the Auto-Blend Plus feature, which ensures that mobile phase compositions and gradients are automatically blended to achieve the optimum retention and selectivity requirements. The Auto-Blend Plus feature automatically manages pH and ionic strength requirements for the mobile phase by calculating the proportions of buffer stocks required for desired conditions. Computation can be based on known p

 values or on an empirical calibration table, making any possible buffer combination available. Users also can create their own library of combinations. Figure 1 shows an example of the H-Class system using autoblend for the analysis of a protein mixture. 

Figure 1: A mixture of proteins separated by cation-exchange chromatography using the Waters Acquity UPLC H-Class Bio system. The Auto-Blend Plus feature was used to automatically find the optimum concentration of mobile phase modifier.			

The NanoAcquity UPLC System with 2D Technology uses two-dimensional (2D) UHPLC for better chromatographic resolution of complex proteomic samples by using a dual reversed-phase approach, which results in better protein identifications, quantification, and sequence coverage. The 2D system uses reversed phase at pH 10 in the first dimension, followed by reversed phase at pH 2 in the second dimension for results that far exceed those of conventional ion-exchange methodologies. 

The NanoAcquity UPLC system with HDX Technology uses UHPLC separations and high-resolution MS for the analysis of biotherapeutics and proteins, and for the routine determination of changes in protein conformation, including drug binding, product stability, and protein interactions. By carefully controlling temperature-sensitive parts of the HDX workflow, conformational changes are not missed. The separation takes place within the cooled environment with the added advantage of sub-2-µm particles for enhanced chromatographic resolution. Maintaining temperatures at 0 ± 1 °C means that back-exchange is kept to an absolute minimum. Once the hydrogen deuterium exchange reaction has been chemically quenched, the HDX Manager directs the sample to a pepsin column for peptic digestion, or directly into the MS system for global analysis. The sample is then trapped for a rapid UHPLC analytical separation. 

Agilent Technologies (Santa Clara, California) introduced two systems with applications in biologics, the 1260 Infinity Bio-inert Quaternary LC and a new mAb-Glyco Chip Kit for the company's 1260 Infinity HPLC-Chip/MS system. The 1260 Infinity Bio-inert Quaternary LC system is based on the 1200 Infinity Series LC platform. The system is 100% inert with completely metal-free capillary surfaces in the sample flow path and no stainless steel (for example, no nickel-, chromium-, cobalt-, or manganese-containing alloys or other alloys that are problematic for biological samples) in the mobile phase flow path. It has a wide pH range (1–13) and can easily tolerate 2 M salt and 8 M urea. Options include a bioinert fraction collector, fluorescence detection, and column- and solvent-selection capabilities. 

The Agilent 1260 Infinity HPLC-Chip/MS System is a microfluidic chip-based technology for nanospray LC–MS introduced in 2005. It combines nanoflow HPLC columns, connecting capillaries, and an electrospray emitter into a re-useable, credit card-size device allowing nano LC–MS to be accessible to scientists without the troublesome setup involving the microvalves, fittings, and capillary tubing of conventional nano LC. With the Pittcon 2011 introduction of the new mAb-Glyco Chip kit, Agilent now offers 12 versions of HPLC-Chips for multiple applications. The mAb-Glyco Chip includes an enzyme reactor for on-chip deglycosylation of mAbs, enrichment and analytical columns for concentration and separation of the cleaved N-glycans, and an electrospray tip. The system includes predefined optimized methods for HPLC-Chip/MS analysis, automated comprehensive data processing tools (including an accurate mass glycan database for ease of characterization), reporting templates, and all reagents and chemicals required. 

Dionex (Sunnyvale, California) introduced the UltiMate 3000 mAb Analysis Platform, which is a fully biocompatible system that features a dual ternary gradient pump design in a 2-in-1 system concept. The system's autosampler includes an integrated fraction collector with the ability to automatically reinject samples into a second dimension. Using the system, cell culture fluid samples containing antibodies can be injected onto a protein A column for antibody recovery and the purified antibody samples can be injected onto an SEC column for aggregate analysis and then injected onto an ion-exchange column for charge variant analysis. 

Historically, conductivity monitoring has been used in the HPLC separation of biologics to accurately follow gradient formation. More recently, on-line pH monitoring has gained interest in the emerging application of pH gradient ion-exchange chromatography of monoclonal antibodies and charge variants. An introduction by Dionex featured the new PCM-3000 module that allows on-line pH and conductivity monitoring in these types of HPLC applications. The module mounts to the UltiMate 3000 UV absorbance detector and features flow cells for analytical LC. 

Shimadzu Scientific Instruments (Columbia, Maryland) introduced the Perfinity Workstation. This system, which was developed in partnership with Perfinity Biosystems (West Lafayette, Indiana), uses novel and proprietary separation technologies to automate sample preparation and analysis of serum proteins. The system prepares peptides ready for LC–MS analysis directly from serum within 10 min. The system consists of five columns; each column performs one step of the sample preparation process: affinity selection, buffer exchange, digestion, desalting, and reversed-phase separation. Automated integration of these steps makes the overall analysis up to 50 times faster than conventional approaches. Standard immunoassays have been the dominant method of performing routine serum protein analyses for the last 60 years for good reason; however, proteins that vary in biological activity are impossible to discriminate using standard antibody approaches. Although LC–MS is capable of resolving these differences, proteins must be extracted or samples fractionated before analysis. The system's solution is to pair the selectivity of antibodies with the resolving power of LC–MS. Figure 2 illustrates a comparison of a traditional protein digest to that obtained using the system. 

Figure 2: Comparison reversed-phase chromatography of a protein digest obtained by traditional (top) and Perfinity Workstationâderived (bottom) digests.			

HPLC and UHPLC systems intended for automating method development and facilitating method transfer have also been introduced at recent Pittcons. This year, new systems and technology were introduced by at least two vendors. Waters formally introduced a system, previewed at last year's Pittcon in Orlando, that combines the Acquity UPLC H-Class system with Fusion method development software (S-Matrix Corporation, Eureka, California). Using quality-by-design (QbD) principles, this combination, along with the introduction of Waters' Method Development Chemistry kits, helps to develop more robust analytical methods in a fraction of the time it takes using traditional HPLC, taking automated method development to entirely new levels of efficiency and productivity, and reducing costs.  

Instrument-to-instrument method transfer is an important topic for many laboratories, where HPLC and UHPLC methods are transferred between different departments or locations with different LC instruments. Agilent Technologies chose Pittcon 2011 to introduce Intelligent System Emulation Technology (ISET) for the 1290 Infinity LC system. This feature harnesses the performance of the 1290 Infinity LC to emulate other systems for the seamless transfer of methods between LC systems, regardless of brand. ISET is designed to accelerate and facilitate the transfer of analytical LC methods between laboratories using different HPLC or UHPLC instruments. Users can take full advantage of the speed and resolution of the newest UHPLC approaches while still running their legacy HPLC methods. This dual-capability means that users can safely invest for the future without disrupting analytical workflows, even in regulated environments. LC method development laboratories can be more productive by taking advantage of faster UHPLC techniques and transferring methods to HPLC by emulating that instrument. 

Agilent Technologies also introduced two new valve head kits for the 1290 Infinity Valve Drive, the latest addition to the 1290 Infinity LC system. It is compatible with all currently available Agilent Quick Change Valve heads and Agilent 1220, 1260, and 1290 Infinity LC systems, as well as with older 1100 and 1200 Series LC systems, facilitating a multitude of column- and solvent-selection configurations for method development. 

With these two new kits, Agilent now has 12 different valve head kits for flexibility in HPLC and UHPLC automation. The first kit is a 1200-bar, six-column selector valve head kit for use with the 1290 Infinity thermostated column compartment that supports up to four columns, a waste line, and a bypass line. Used together with the new 1290 Infinity Valve Drive, as many as six columns are supported (the limitation inside the column compartment is because of available presolvent heat exchangers). Together with this latest addition to the valve portfolio, Agilent customers can decide between two-, four-, six-, or eight-column selection configurations in an LC system by a simple mouse-click, depending on the valve head used. The second kit is a 12-position selection bioinert valve head kit. Typically, this kit will be used with the 1290 Infinity Valve Drive for solvent selection or fractionation at low pressures (up to 200 bar). 

Charged aerosol detection (CAD) was first introduced commercially in 2004 and is based on the combination of HPLC with electrical aerosol technology. CAD is highly sensitive, provides a consistent response, and has a broad dynamic range, offering real advantages to researchers and analysts for a variety of applications, particularly when analyzing compounds lacking UV chromophores.  

Introduced this year, the Corona ultra RS is the newest Dionex CAD system. This version adds features that enhance the usability and extend the applications for the Corona ultra detector. The RS version includes an on-board switching valve that allows the detector to be used independently of other detectors, without the need for system reconfiguration. The same valve can be used to divert high salt from samples. An optional variable ratio flow splitter is available to make it easier to use CAD in parallel with MS without compromising resolution, and a new data processing algorithm makes it possible to optimize the data output for specific applications. The detector is fully compatible with both HPLC and UHPLC methods, and when used with the company's UltiMate 3000 dual gradient system, the detector provides consistent response independent of the molecule's chemistry for nonvolatile analytes with or without a chromophore. 

Triple-quadrupole MS is the method of choice for accurate quantification and confirmation of trace-level analytes in complex matrices, from the detection of drugs and metabolites in biological specimens to environmental contaminants and pesticides in food. Keeping pace with the sensitivity and speed demanded by today's HPLC and UHPLC separations, Shimadzu introduced the LCMS-8030 system, which couples the power of a triple-quadrupole MS system that claims industry-leading fast multiple reaction monitoring (MRM) transitions (500/s), fast polarity switching, (15 m/s), and scan speeds of 15,000 u/s. 

Dionex expanded its ion chromatography (IC) offerings with several Pittcon 2011 introductions. The ICS-5000 High Pressure Capillary IC system allows analysts to increase throughput and lower costs by conducting fast IC separations, or to achieve higher separation efficiencies through the use of longer columns. The new capillary version of the system can operate continuously at pressures as high as 5000 psi. Also introduced was the AS-AP IC autosampler for capillary IC and fast IC applications. The autosampler has options for automatic dilution and an integrated sample conductivity and pH accessory; concentration and matrix removal applications, along with low carryover and a contamination-free flow path, make it ideal for ultratrace ion analysis. It also supports dual IC systems, fraction collection, and sample preparation. The autosampler offers fast cycle times and high sample capacity, from 81 samples with 10 mL vials to 1152 samples using microwell plates, supporting fast IC and providing the needed capacity for high-throughput laboratories. The chemically inert PEEK flow path withstands pH extremes and protects sensitive samples from metal contamination. Figure 3 shows separations obtained using the ICS-5000 High Pressure Capillary IC system. 

Figure 3: Fast determination of a 19-anion standard using a Dionex High Pressure Capillary IC system. An IonSwift MAX100G, MAX100, 0.25-mm column operated at 35 Â°C and a KOH gradient was used. The injection volume was 0.4 ÂµL, and detection was by suppressed conductivity. Peaks: 1 = fluoride, 2 = acetate, 3 = formate, 4 = butyrate, 5 = galacturonate, 6 = chloride, 7 = nitrite, 8 = bromide, 9 = nitrate, 10 = carbonate, 11 = sulfate, 12 = oxalate, 13 = tungstate, 14 = phosphate, 15 = chromate, 16 = citrate, 17 = isocitrate, 18 = cis-aconitate, and 19 = trans-aconitate.			

In addition, Dionex introduced the SRD-10 suppressor regenerant detector and the SCC-10 suppressor current controller. The suppressor regenerant detector is a stand-alone device that monitors liquid flow to a suppressor and disables the eluent pump if flow is restricted or stops, preventing irreversible damage to the suppressor.  

The suppressor current controller is an external adapter designed for use with legacy instruments that offer only four settings for electrolytic suppressor current. The controller's output current can be set in 12 discrete steps from 10 to 250 mA, thus increasing suppressor life while simultaneously reducing noise. It also provides greater flexibility in selecting optimal operating conditions and suppressor current settings for many IC applications. Because the controller connects the suppressor's power supply to the suppressor, and because it is powered from the existing suppressor power supply, it is not necessary to change the system configuration in the control software for use. 

To address the needs of process development chemists and QbD, Waters introduced the Patrol UPLC Laboratory analyzer, an integrated system solution designed and engineered to perform laboratory and pilot-scale online reaction monitoring in the process development laboratory in a fully automated and compliant-ready manner. Based on the company's UPLC platform, it is designed to detect and quantify the components of complex reaction mixtures and fully characterize a candidate molecule by LC, LC–MS, and LC–MS-MS before moving it into process development. The analyzer enables process development scientists to directly analyze the progress of reactions online by Real-Time LC or LC–MS to generate quantitative results, including low-level reaction components and trace process impurities. A hallmark of the system is its dilution accuracy, dilution linearity, and dilution range. The technology behind this performance is the instrument's process sample manager (PSM), which automatically extracts an online sample from a reactor or slipstream, and performs sample preparation and sample injection — a process that once required the time and full attention of an analytical technician. In addition to online samples, the PSM can also accept and store up to 32 barcode-labeled vials, which can be a combination of different standards, controls, and even at-line samples.  

Several new software introductions were made at Pittcon this year, including laboratory information management systems (LIMS) software, chromatography data systems (CDS), and extensions of existing software to extend control capabilities. 

Thermo Fisher Scientific (Waltham, Massachusetts) launched a significant release of the company's flagship laboratory data management software, which is designed for large-scale process and industrial laboratories and is developed to facilitate compliance with ISO and GLP. Thermo also showcased LIMS software for water and environmental applications, designed to help laboratories keep pace with the rapidly changing regulatory environment, including the Safe Drinking Water Act and the EPA's Drinking Water Infrastructure Needs Survey and Assessment. 

Waters debuted the next version of its popular Empower product, the Empower 3 chromatography data system, which is a scalable, enterprise-wide CDS platform that can be rapidly deployed and fit easily into existing corporate infrastructure. The software is designed to address the needs of technology managers for data management standardization, including reduced training efforts and support resources, improved internal communication and information exchange, and the facilitation of more-consistent regulatory compliance through easier software validation and streamlined review and sign off of results. In addition, the software offers a variety of usability and workflow enhancements that help streamline users' daily workflow and efficiency. The software features improved navigation between the review and reporting applications and also allows users to create sample sets after acquisition. This feature enables more flexibility in processing injections that were acquired individually and also helps with multi-stage and delayed-release dissolution testing. Additional calculation capabilities allow the ability to determine current signal-to-noise requirements, according to regional pharmacopeias in the United States, European Union, and Japan. 

Another unique feature of the CDS software is the capture of Acquity UPLC column usage information, including information such as column name, serial number, the number of injections on that column, maximum pressure, and maximum temperature. This information is tracked for each sample set for automatic and accurate documentation purposes; it is also tracked on a rolling basis so users can obtain column trending information and extrapolate and predict lifetime information. 

In the area of sample preparation, Dionex announced Chromeleon CDS control of its ASE 350 automated solvent extraction system. The control capabilities feature control of chromatographic systems, creation of secure audit trails, and hard copy reports not available from standard front panel controls. 

Supercritical fluid chromatography (SFC) has been commercially available since around 1982. SFC can be differentiated from gas chromatography (GC) and HPLC by the use of a supercritical fluid as the mobile phase.  

SFC has several advantages over GC and HPLC, including rapid separations without the use of organic solvents, reduced use of organic additive chemicals, and the use of carbon dioxide collected as a by-product of other chemical reactions (or collected directly from the atmosphere), contributing no new chemicals to the environment. In addition, SFC separations can be done faster than HPLC separations because the diffusion of solutes in supercritical fluids is about 10 times greater than that in liquids (and about three times less than in gases). The lower diffusion results in a decrease in resistance to mass transfer in the column and allows for fast high resolution separations. Compared with GC, capillary SFC can provide high-resolution chromatography at much lower temperatures. This allows fast analysis of thermolabile compounds. Many vendors have recognized these advantages and have begun to offer SFC instruments on existing HPLC and UHPLC platforms. 

The new Agilent 1260 Infinity Analytical SFC system uses the Aurora SFC Fusion A5 (Aurora SFC Systems, Inc, Redwood City, California) module to achieve high sensitivity to facilitate low-level impurity analysis as well as enantiomeric excess determination, in chiral compound separations. It is available as a complete system or as an upgrade to existing Agilent 1100 and 1200 Series or 1260 Infinity LC systems. Four orders-of-magnitude of dynamic range provide accurate quantitation of 0.01%-level impurities with high precision, HPLC-like sensitivity, and UHPLC speed using UHPLC columns. The system is fully compatible with the manufacturer's CDS software. Figure 4 illustrates the separation of a chiral compound with two chiral centers using the Agilent 1260 Infinity SFC LC system. All four stereoisomers were baseline separated isocratically. 

Figure 4: Isocratic SFC separation of four stereoisomers of a chiral pharmaceutical compound with two chiral centers using an Agilent 1260 Infinity SFC LC system.			

Waters also introduced an SFC system, the Acquity UPSFC system, which is built on the company's UPLC sub-2-µm particle chemistry platform. The UltraPerformance supercritical fluid chromatography (UPSFC) system is designed to incorporate all of the advantages of SFC, in addition to superior separation, resolution, and speed afforded by the sub-2-µm particle chemistry platform. When used in chiral and achiral compound applications, the sensitivity and separation capability of the system enables users to rapidly confirm the purity of compounds and to determine enantiomeric excess. Compared to traditional analytical SFC, the sub-2-µm particle system can run more samples per day with shorter cycle times, faster re-equilibration, and higher resolution. The system is fully compatible with the company's Empower and MassLynx informatics software and with the company's Viridis SFC Columns that are specifically designed for SFC. 

The information in this review is based partially on manufacturers' responses to a preconference questionnaire mailed in late 2010. I used the information received in the questionnaires that were returned, as well as information from personal communication, to try to make this review as comprehensive as possible. But keep in mind that, because of the sheer size of the conference and the fact that some manufacturers did not respond to the questionnaire or do not release pre-show information, this report cannot be considered an exhaustive listing of all new products introduced in Atlanta. I'm sure there were a lot of additional noteworthy items at Pittcon, but time, space, and the scope of this column prevent me from covering them all in detail. I'm bound to have missed a few items or details, and I apologize for any omissions. In 2012, Pittcon will return to Orlando, March 11–16. I hope to see you there! 

 "Innovations in HPLC" Editor Michael E. Swartz, Ph.D., is Principal Scientist in Analytical Development at Ariad Pharmaceuticals, Cambridge,Massachusetts, and a member of LCGC's editorial advisory board. Direct correspondence about this column to "Innovations in HPLC," at 

Innovations in HPLC

This year, many HPLC systems focused on facilitating process development, method transfer, and biological analysis.

HPLC Systems and Components Introduced at Pittcon 2011: A Brief Review

John Hinshaw reviews the new GC instruments and accessories that were on display at Pittcon 2011, which was held this March in Atlanta, Georgia.

The 62nd annual Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (Pittcon), March 13–18, 2011, returned to Atlanta, Georgia, for the first time since 1997. I particularly enjoyed Atlanta for the Southern hospitality and food, easy access to and inside the Georgia World Congress Center, and the peach tree blossoms in Centennial Olympic Park. The conference exhibition floor provided some relief for weary aisle-walkers who took the time to hike to the far corners. There I found at various booths free fresh-baked chocolate chip cookies, coffee, croissants, a printed invitation to discuss the field of "chromatology," a new type of chair for surviving trade shows (free demonstrations offered), the usual superhero costumes, and electrical massage demonstrations that had some of us jumping around like Luigi Galvani's 18th-century frogs. 

Pittcon total attendance in 2011 increased to 17,199 — up about 300 from 2010. The number of exhibiting companies increased slightly too, as did the number of exhibitor booths. Next year, the Pittsburgh Conference will return to the Orange County Convention Center in Orlando, Florida, March 11–15, 2012. If the next winter season is as inclement and extreme as this year's was, then the week's sojourn at this wandering conference's southernmost latitude will be welcomed enthusiastically.  

Overall, 2010–2011 was another strong year for gas chromatography (GC). A number of companies introduced new GC instruments, both for the laboratory and as portable systems, rounded out by a significant number of new and improved automated sampling systems, highperformance mass spectrometry (MS) detectors, and clever time-saving accessories. This year's count of GC products is about even with each of the last two years, which shows that GC remains relatively immune to declining markets while showing significant growth in the high speed, miniature, micro, and portable areas. 

This annual "GC Connections" installment reviews GC instrumentation and accessories shown at this year's Pittcon or introduced during the previous year at conferences such as ASMS or Analytica. For a review of new GC and liquid chromatography columns and related accessories, please see "Column Watch" in the March and April 2011 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 actual use or experience of the author. During the conference, 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 given the preliminary nature of some of the material, 

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

The trend in GC instruments at this year's Pittcon ran toward smaller size, reduced environmental impact, less carrier gas and power consumption, and improved ease of use. A list of some of the new instuments on offer is shown in Table II. 

The newest routine laboratory chromatographs feature high-efficiency oven insulation and smaller oven sizes that yield faster heating and cooling rates while using less power and supporting high speeds of analysis. The smaller mini- and micro-GC systems, by their nature, consume less power and carrier gas in a smaller footprint; several were hand-portable. 

Table II: New GC instrument systems (continued) 

Last year's industry consolidations resulted in a number of new GC instrument announcements from companies such as Bruker (Bremen, Germany), Agilent Technologies (Santa Clara, California), and Thermo Fisher Scientific (Waltham, Massachusetts) based on the models acquired last year.  

One new company, Ellutia Chromatography Solutions (Charleston, South Carolina) announced it was forming by unifying a group of three partner companies: Advanced Chromatography Systems, Cambridge Scientific Instruments, and Unicam Chromatography. All told, there were at least 14 new GC instruments on display — a remarkable assortment for an allegedly mature technology. 

Two new time-of-flight (TOF) MS detectors for GC stood out at this year's Pittcon. Offering the highest mass resolution and speeds in their class for demanding highperformance techniques such as high-speed GC or comprehensive GCxGC, TOF-MS detectors are ideal for analyses in which an MS detector enhances compound identification and quantification beyond the capabilities of the separation system alone, such as in a wide range of environmental, petroleum, food and flavor, and pharmaceutical applications. In the more conventional detector vein, Detector Engineering and Technology (Walnut Creek, California) is offering a new series of their retrofittable thermionic detectors, including a unique catalytic combustion ionization method for the selective detection of methylene functional groups. A list of some of the new detectors on offer is shown in Table III. 

Sampling systems for GC saw increased activity, too. Agilent introduced a significant new development in headspace samplers with the model 7697A. The new design leverages the company's existing liquid auto-sampler's capabilities in a complete redesign of the previous workhorse headspace sampler.  

Thermal desorption and pyrolysis took center stage too, with new products from Frontier Laboratories (Fukushima, Japan), Gerstel (Mülheim, Germany), and CDS Analytical (Oxford, Pennsylvania) along with a new cannister sampling accessory from Markes International (Llantrisant, United Kingdom). The common thread among all of these was automated handling of gaseous samples from diverse sources. Gerstel also introduced an updated version of its MPS multipurpose sampling and sample preparation robot. A list of some of the new GC sampling products on offer is shown in Table IV. 

Two accessories for capillary GC users are listed here. The Restek Flip-Seal (Restek, Bellafonte, Pennsylvania) for Agilent split–splitless inlets assists lower-seal maintenance and includes two useful seals in one part. From NLISIS (Eindhoven, The Netherlands), the Fuse capillary column connector accessory enhances column-to-column or precolumn connections.  

Two new gas regulators were introduced with reduced contamination in mind. The AirGas regulator (AirGas, Radnor, Pennsylvania) includes a new check-valve on the high-pressure side to control tank and stream contamination with air or other gases in the regulator. Concoa (Virginia Beach, Virginia) showed a regulator with inert metal surface treatment to reduce interaction with sensitive sample components. Analytical Flow Products (Thetford Mines, Quebec, Canada) had two designs of rotary gas sampling valve actuators, and both AirGas and Concoa exhibited high-performance, high-pressure, analytical gas delivery manifolds. 

I would like to thank the manufacturers and distributors that kindly furnished the requested information before, during, and after Pittcon 2011, allowing a timely report on new product introductions. For those manufacturers who did not receive a preconference questionnaire this year and would like to receive one and be considered for early inclusion in our Pittcon 2012 coverage, please send the name of the primary company contact, the mailing address, fax number, and e-mail address to Laura Bush, 

, Advanstar Communications, 485F US Highway One South, Suite 100, Iselin, NJ 08830, or e-mail 

"GC Connections" editor John V. Hinshaw is Senior Scientist at BPL Global, Ltd., Hillsboro, Oregon, and a member of LCGC's editorial advisory board. Direct correspondence about this column to the author via e-mail: 

GC Connections

This was a strong year for GC systems. Here's what is new.

New Gas Chromatography Products at Pittcon 2011

An effective workflow has been established that uses chiral as well as achiral stationary phases in combination with compound-specific gradient methods for fast mass-directed supercritical fluid chromatography (SFC) purification of complex mixtures. It has been used successfully to purify mixtures that show little or no separation in reversed-phase high performance liquid chromatography (HPLC).

Supercritical fluid chromatography (SFC) has been considered a state-of-the-art chiral separations technique for the past decade (1–5), while recently emerging as a choice technique for achiral separations (6,7). The primary driving force for this operational movement is the commercialization debut of mass-directed preparative units, which adopt similar hardware and software as the widely used reversed-phase high performance liquid chromatography (HPLC) system (8,9). Beyond their normal-phase chromatographic capabilities, SFC systems fulfill the need for speed and efficiency with numerous practical advantages relative to reversed-phase HPLC including higher throughput, shorter cycle times, and lower operating costs because of the "green" nature of these systems that results from lower solvent consumption and waste disposal (10–12). 

Figure 1: Analytical screening of stationary phases Chiralpak IA, IB, IC, and achiral 4-ethylpyridine. Gradient: 10â60% modifier in 5 min; flow rate: 4 mL/min. Starburst symbol highlights the peaks of interest. 

Despite the current expanding application of SFC in the discovery laboratories of the pharmaceutical industry, only a limited number of interesting approaches for difficult achiral SFC purification have been reported (13–15). No standardized methods or generic workflow appear to have been adopted. Experiences at our drug discovery laboratories have shown that the implementation of a generic workflow, with the appropriate combination of the mobile phase and a collection of selected stationary phases, can significantly reduce the turnaround time of purification and thus increase efficiency. For this purpose, a special workflow was developed that consists of an effective and fast-screening protocol using chiral and achiral stationary phases in combination with compound-specific gradient methods for fast mass-directed SFC purification of complex mixtures (16). It has been successfully used for the purification of structurally similar diastereomeric and regioisomeric mixtures that have little or no separation in reversed-phase HPLC. With faster separation, higher sample throughput, less organic solvent usage, and mass-triggered fraction collection, the SFC–mass spectrometry (MS) technique has become a primary purification tool for difficult achiral samples in our laboratories. 

Figure 2: Preparative profile obtained using a Chiralpak IA column (gradient: 40â60% modifier in 18 min; flow rate: 30 mL/min) and QC of purified products. 

 The analytical and preparative SFC–MS systems used were from Thar Technologies (Pittsburgh, Pennsylvania). 

The analytical SFC system consists of a Thar SFC front end with a total flow rate up to 10 mL/min, a Waters ZQ mass detector (Waters Corporation, Milford, Massachusetts) and six columns, including Chiralpak IA, IB, IC (Chiral Technologies Inc., West Chester, Pennsylvania), 4-ethylpyridine (Princeton Chromatography Inc., Cranbury, New Jersey), Chromegabond Pyridine Amide (ES Industries, West Berlin, New Jersey), and CN (Zymor, Inc., Wayne, New Jersey). The system is operated using Masslynx software from Waters. 

Table I: Separation factor at analytical level 

Rapid SFC analytical screening was performed primarily on chiral stationary phases (CSPs) and occasionally on achiral stationary phases when chiral columns failed to achieve resolution of the analytes. The rationale behind this approach was based on our empirical data generated from compounds purified over the past two years. Polysaccharide-based CSPs (100 mm x 4.6 mm, 5-µm columns) and achiral alkyl bonded stationary phases (150 mm x 4.6 mm, 5-µm columns) were screened in the following order: IA, IB, IC and 4-ethylpyridine, Chromegabond Pyridine Amide, CN. Only polysaccharide-bonded CSP columns were screened because of their high compatibility with a broad range of sample solubilizing solvents (such as dimethyl sulfoxdide [DMSO] and dimethylformamide [DMF]) (3). 

Figure 3: Analytical LC (column: 100 mm x 2.1 mm, 1.7-Âµm BEH C18; gradient: 5â90% acetonitrile in 1.5 min; flow rate: 0.6 mL/min) and SFC chromatograms of the mixture (columns: 100 mm x 4.6 mm, 5-Âµm Chiralpak IC; gradient: 10â60% modifier in 5 min; flow rate: 4 mL/min).  

The analytical screening was performed with a gradient of 10–60% in 5 min and a modifier of 0.2% isopropylamine in methanol. Because most drug discovery programs produce compounds that are basic or halogenated (or both) in nature, improved peak shape and enhanced loading of compounds may be achieved using amine additives instead of pure modifiers. Based on our experience, 0.2% isopropylamine additive in methanol was identified as the best modifier system in terms of resolution, after screening a variety of amines and different concentrations of isopropylamine (data not shown). Experimental results showed that increased concentrations of isopropylamine did not yield additional improvement of the chromatographic resolution, but instead had a damaging effect on the ionization pattern. The flow rate was 4 mL/min, the back pressure was set at 150 bar, and the column temperature was kept at 40 °C. 

Figure 4: Preparative profile obtained using a Chiralpak IC column. Mobile phase: isocratic, 25% modifier; run time: 16 min; flow rate: 30 mL/min. 

Preparative SFC–MS runs were performed on a Thar SFC–MS Prep 30 system; a fully integrated MS directed preparative SFC unit, equipped with a Waters 3100 mass detector, a Waters 2757 sample managing module for open bed fraction collection, and a Thar SFC front end with a total flow rate of up to 30 mL/min, using various 250 mm x 21 mm preparative columns. Samples injected into the system were dissolved in DMSO or DMF. A compound-specific preparative SFC gradient was derived from the retention time and elution profile of the compound in the analytical run (3). The flow rate used was 30 mL/min and additional chromatographic conditions used in the preparative purification included a back pressure of 150 bar, a column temperature of 40 °C, and 0.2% isopropylamine in methanol as modifier. Injection volumes ranged from 100 to 500 µL with an initial crude sample concentration of 50–70 mg/mL. Mixtures used in this study were all synthesized and submitted to purification by our internal medicinal chemists. 

Figure 5: Analytical LC (column: 100 mm x 2.1 mm, 1.7-Âµ1m BEH C18; mobile phase A: acetonitrile; mobile phase B: 0.01% formic acid in water; gradient: 10â90% A in 1.5 min; flow rate: 0.6 mL/min) and SFC chromatograms of the mixture (columns: 150 mm Ã 4.6 mm, 5-Âµm Chromegabond Pyridyl Amide; modifier: 10% of 0.2% isopropylamine in methanol; flow rate: 4 mL/min).  

 The first example involved the purification and separation of a diastereomeric mixture. The differences in selectivity of the stationary phases are highlighted in Figure 1. For this specific mixture, the Chiralpak IA column demonstrated the best separation of the diastereomers, in addition to other components, in the crude mixture. Although the Chiralpak IC column also showed suitable separation of all constituents, the Chiralpak IA column was chosen for preparative purification based on its higher column loading and better chromatographic performance. A compound-specific preparative SFC gradient of 40–60% in 4 min and flow rate of 30 mL/min was subsequently applied and successfully isolated the desired compounds from the impurities (Figure 2). The injection volume used here was 200 µL with a sample concentration of 50 mg/mL in DMSO. Following the purification, the overall purity of the collected product fractions was confirmed by performing an SFC–MS analysis. The purification was successful in isolating the two isomers with purities by UV

Figure 6: SFC preparative profile obtained using a Chromegabond Pyridyl Amide column (mobile phase: isocratic, 25% modifier; run time: 14 min; flow rate: 30 mL/min) and QC of products using LC. 

 The second example involved the purification and separation of a complex four-component mixture of regioisomeric pairs with their corresponding methylated impurities (+14 Da). The mixture was poorly resolved via LC–MS. However, an analytical SFC–MS screening workflow identified Chiralpak IC as a feasible stationary phase for the separation of the mixture (Figure 3, symbol highlights the peaks of interest). Mass-directed preparative SFC purification under 25% isocratic conditions successfully isolated the two regioisomers, as well as their methylated by-products (Figure 4). The injection volume was 100 µL with a sample concentration of 200 mg/mL in DMSO. 

Figure 7: Analytical LC and SFC chromatograms of the mixture on Chiralpak IA. LC column: 50 mm X 4.6 mm, 3.5-Âµm XBridge C18; gradient: 5â90% acetonitrile in 2 min; flow rate: 1.0 mL/min. SFC column: Chiralpak IA; gradient: 10â60% modifier in 5 min; flow rate: 4 mL/min. 

 The third example involved the purification and separation of a 

-isomeric mixture. The differences in separation factor on LC–MS versus SFC–MS screening phases are highlighted in Table I. For this specific sample, the Chromegabond Pyridyl Amide column demonstrated the best separation conditions for resolving the mixture (Figure 5). The performance of preparative SFC–MS separation with a 150 mm x 21 mm Chromegabond Pyridyl Amide column under 25% isocratic conditions with a flow rate of 30 mL/min and a back pressure of 150 bar is shown in Figure 6. Following the purification, the overall purity of the collected product fractions was confirmed by LC–MS analysis. The successfully isolated 

-isomer had a purity of ~99.99%, while the 

-isomer had a purity of 94.16%. It is important to note from experimental results that the 

-isomers displayed reversed elution order on achiral columns in SFC versus reversed-phase HPLC, while the same elution order (data not shown) was observed on chiral columns for both SFC and reversed-phase HPLC (16). 

Figure 8: Preparative profile obtained using a Chiralpak IA column. Mobile phase: isocratic, 20% modifier; run time: 18 min; flow rate: 30 mL/min. 

 The fourth example involved the separation of a very polar product mixed with its methylated impurity (+14 Da), which tends to be coeluted in reversed-phase HPLC. The initial desired product purity was approximately 84.66% from a typical mass-directed HPLC purification. A higher purity of 99.8% was needed for in-vivo pharmacological evaluations and both target and impurity needed to be isolated and characterized. For this specific crude mixture, the Chiralpak IA column demonstrated the highest resolving power (Figure 7). Mass-directed preparative SFC purification performed under the isocratic conditions (20% modifier in 18 min, flow rate 30 mL/min) successfully isolated the desired compound from its methylated impurity (Figure 8). The final recovery for the desired product was 85% with a purity of 99.9% by HPLC (Figure 9). The injection volume used here was 400 µL with the crude sample concentration of ~70 mg/mL. 

Figure 9: HPLC chromatogram of the mixture and QC of the purified products. Column: 50 mm X 4.6 mm, 3.5-Âµm XBridge C18; gradient 5â90% acetonitrile in 2 min; flow rate: 1.0 mL/min. 

Based on our experiences, the SFC gradient screening strategy delivers fast method development for the resolution of complex achiral mixtures, which enables their translation to preparative SFC. When comparing chiral to achiral columns, the chiral columns provide a higher success rate in terms of recovery and purity of the purified products from isomeric purification, making them more efficient and therefore offsetting associated higher costs. Those benefits have been confirmed for all isomers purified by our laboratory over the past two years. 

To the best of our knowledge, this is the first effective workflow that combines short chiral stationary phases with fast gradients for rapid SFC purification of pharmaceutical achiral compounds. It should be applicable to the purification of a wide number of pharmaceutically relevant compounds. 

(3) G. Felix, A. Berthod, P. Piras, and C. Roussel, 

(6) R.T. McClain, A. Dudkina, J. Barrow, G. Hartman, and C. J. Welch, 

(8) V. Lazarescu, Y. Li, M.J. Mulvihill, and L. Ma, poster presented to the 2nd International Conference on Packed-Column SFC in Zurich, Switzerland, October 1–2 (2008). 

(10) T. Berger, K. Fogleman, T. Staats, P. Bente, I. Crocket, W. Farrell, and M. Osonubi, 

(11) M. Ventura, W. Farrell, C. Aurigemma, K. Tivel, M. Greig, J. Wheatley, A. Yanovskya, K.E. Milgram, D. Dalesandro, R. DeGuzmana, P. Tran, L. Nguyena, L. Chunga, O. Grona, and C. A. Koch, 

(12) W.C. Ripka, G. Barker, and J. Krakover, 

This process can improve efficiency in a drug discovery laboratory.

LCGC North America-05-01-2011

Self-Assembled Nanomaterials for Enhanced Chemical Separations

Ghost Peak Investigation in a Reversed-Phase Gradient LC System

HPLC Systems and Components Introduced at Pittcon 2011: A Brief Review

New Gas Chromatography Products at Pittcon 2011

A Generic Workflow for Achiral SFC Purification of Complex Pharmaceutical Mixtures

Establishing USP Rebaudioside A and Stevioside Reference Standards for the Food Chemicals Codex

Vol 29 No 5 LCGC North America May 2011 Regular Issue PDF