Keynote Presentation

Frantisek Svec
Department of Chemistry, University of California, Berkeley, CA 94720-1460

"New Selective Phases for HPLC Column Technology"

In recent years, there have been many advances in HPLC column technology, which in turn have yielded a wide range of new products. These advances are reflected in both the base silica and in the stationary phase chemistry. The modern arsenal of columns now includes base deactivated, pH stable phases, small particle packings, alumina-based phases, and of course, the standard C8, C18 and Phenyl materials.

It is the goal of this Column Development Seminar to help unravel and provide practical information on many of the new developments in HPLC column chromatography. The presentation contains information on several column chemistries including perfluorinated stationary phases; pH stable polymer coated alumina, and reversed phase columns for highly aqueous mobile phases. The development and application examples will be provided for each of these stationary phases, as well as use guidelines. The seminar will also include a discussion on the reproducibility and column durability for these new phases.

"The ATAS Optic2 GC Injector"

Authors: G. P. Brown

Affiliation: Giangario Scientific Co., Inc.

The Optic2 is a rapid, large volume, temperature and pressure programmable injection system suited for use with all common GC and GC-MS systems. The potential for expanded and improved results using the Optic2 is illustrated with examples from the literature. The features of the Optic2 make possible the analysis of compounds up to about 1500 Daltons, the analysis of thermally desorbed volatiles from solids loaded into the injector, et.al. The control and programmability functions allow incorporation into robotic sample preparation, derivatization and analysis systems.

"OOPS - I should have read the manual."

Author: Ed Dietz

Affiliation: OxyChem

ABSTRACT

Chromatographers with years of experience "know it all" and seldom need to read instruction manuals or tip sheets when doing a "simple" analysis - right? Well, this isn’t exactly true as I will show by tracing the history of a gas chromatography problem I recently experienced. What was going to be a minimal effort study to determine several impurities in methanol, turned out to be a frustrating and embarrassing effort to explain why peak shapes and retention times randomly exhibited minor to very major changes. Follow me through my testing and problem diagnosis methods and see how soon you can explain my operating error.

As we are about to embark into the new millennium, it is worthwhile to reflect on where we stand today and where HPLC column technology may be heading in the future. In this paper, I will briefly discuss the progress achieved in HPLC columns through the 1990’s and speculate where column technology may change. The major improvements in HPLC columns have occurred in four areas: quality, performance, lifetime and design. Manufacturers, driven by user needs and ISO 9000, EN45001, GLP, cGMP and other quality or validation requirements, have made great strides in achieving better column-to-column reproducibility. When coupled to an improved user understanding of limitations and potential misuses of silica-based bonded phase columns, column reproducibility is today of less concern than it was a decade ago. Newer, more stable bonded phase materials capable of going to higher pH values, increased use of guard columns and other column-protecting devices and columns based on highly purified spherical silica particles with few residual silanol groups have provided increased column lifetimes and minimal tailing, particularly with basic compounds. Improved sample preparation technologies such as solid phase extraction, supercritical fluid extraction, and newer generation solvent extraction devices have permitted injection of "cleaner" samples further extending lifetime. Polymeric materials with higher efficiency available in a wider variety of stationary phases are coming into mainstream use, particularly in bioscience and ion chromatography applications. In order to achieve better separations and faster analysis times, chromatographers now favor smaller particle packed columns with 5-um being the standard compared to 10-um a decade ago. The increasing use of 3-um materials packed into shorter columns will undoubtedly make them the standard "workhorse" columns in the future.

In terms of column design, microbore and fused silica-packed columns, that have never realized their earlier potential, are gaining a renewed interest. The increased scrutiny in laboratory budgets, particularly in solvent purchase and disposal costs, required higher sensitivity with smaller samples, and availability of newer generation instruments capable of optimum performance with these narrower bore columns have been driving forces in reconsideration of these columns. The strong interest in high throughput analyses, combinatorial chemistry, drug-candidate screening and LC/MS and LC/MS-MS has led to the need for very short columns packed with small diameter packings and this trend will continue to affect column design into the new century. New analytical column designs such as integrated guard columns, high pressure glass, glass-lined, PEEK, titanium and plastic construction have given the chromatographer a wider choice of inert materials, especially important for biocompatibility and trace inorganic analysis. New phases such as the monoliths and molecular-imprints may revolutionize the way that column technology may evolve. This presentation will cover these aspects of column development and speculate on future direction of column technology in the coming years.

HPLC columns with particle sizes under 5 µm, and with dimensions up to ten-times smaller than traditional analytical-size columns are in increased use. While these column configurations have been possible for many years, their acceptance was needed to stimulate their widespread use in combinatorial chemistry (combichem) and in general where fast separations are useful. As an offshoot of need for faster separations, preparative-scale (1" diameter) short columns (50-150mm) have been developed-allowing rapid separations with larger sample loads. The trend toward smaller column configurations (1 mm i.d. columns and (1.5 µm particles) continues, but practical considerations may limit the chromatographer's ability to obtain expected results for a particular column configuration and sample. Practical issues will be addressed within the context of ultra-fast separations (e.g., < 1 minute) using current 3.5 and 5 µm totally porous silica technology. 'Ultra-fast' separations (e.g., <10 min) will be shown for analysis of more-complex samples (peptides/proteins and anthocyanins).

"Compatibility of Reversed Phase HPLC Columns with Pure Aqueous Mobile Phases"

This study describes various reversed phase HPLC columns according to their stability and reproducibility in pure aqueous mobile phase and provides some possible explanations for the different retention characteristics observed among various columns and the mechanisms that may be involved. One common explanation for this phenomenon of retention loss is that the hydrophobic alkyl chains of the stationary phase fold over when exposed to pure aqueous mobile phase, greatly reducing their interaction with the analytes. Methods for avoiding and reversing the process of retention loss will be presented. New specialty bonded phases that are immune to this phenomenon generally contain some polar character. Polar groups embedded in the alkyl chain can provide stability in pure aqueous conditions but may also cause changes in selectivity. An alternative approach to introducing polar character to the stationary phase that increases retention of polar analytes while maintaining a selectivity that is very similar to that of a conventional C18 column will be shown.

"XTerra™ Columns: High Speed – High Temperature – High pH"

Authors:

Affiliation: Waters Corporation

ABSTRACT

A chromatographic separation was developed for several components. Solid phase preparation of the biological samples culminated in two eluates, which could not be recombined due to endogenous interferences. Rather than dedicating two of our four laboratory’s chromatographic systems, or investing further extensive development time, we employed a single chromatographic system using tandem column analysis with switching valves. Use of two internal standards, one for each phase of elution, was necessarily incorporated. This design allowed us to recombine an individual sample’s chromatographic fractions into a single chromatogram. Use of similar techniques can allow for cost-effective and efficient methods.

Major advances have been made over the past three years in the development of fast generic HPLC methods in support of drug discovery. Through the use of short (30 - 50 mm), narrow bore columns packed with small particle sizes (3 - 5 µm), researchers have been able to reduce the chromatographic run times to 3 - 5 minutes. However, even with the concurrent advances in instrumentation, only approximately 20 % of the compounds in a combinatorial array are analyzed. There continues to be a need to make further improvements in sample throughput.
The 3 -5 minute runtimes that are typical today have been achieved through a reduction in the gradient time, t_g, with a concurrent decrease in column length, L, diameter, d, and an increase in the column flow rate, F. This approach, while proving effective, has limitations. Reductions in column length and increases in flow result in a loss in efficiency and it is this loss in efficiency that ultimately limits the resolving power. To achieve further reductions in run times, while maintaining resolution, requires shorter columns that are optimized for the highest efficiencies at these very high flow rates. This is the promise held by 2 µm particles.
In this poster, we will demonstrate the superior resolving power of 2 µm particles in short columns compared to their larger particle counterparts due to their higher column efficiencies per unit length.

"Strategies for Fast LC/UV and LC/MS Method Development"

Pharmaceutical scientists face significant challenges when trying to develop LC/UV and LC/MS methods rapidly in order to support the drug discovery and development process for new drug candidates. A key step in meeting these challenges is the proper selection of the "best" reversed-phase HPLC column. This column must satisfy the resolution requirements of the method, deliver reproducibility for ruggedness and robustness, and provide performance characteristics that simplify the development process.
In this paper, we will demonstrate how a sound method development strategy combined with the highest quality HPLC columns accelerates the method development process. The critical parameters affecting selectivity and reproducibility of reversed-phase columns within a very short period of time will be discussed in great detail. The latest advances in bonded-phase technology utilizing an organic/inorganic hybrid porous particle technology that offer unique selectivity will be presented. The methylsiloxane incorporated in this organic/inorganic hybrid packing material reduces the surface silanol activity dramatically. This reduced silanol activity improves the peak shapes and also facilitates the method development for a wider pH range (pH 2 to pH 10).  

Solid-phase extraction (SPE) methods are widely used for the extraction of drugs from biological fluids (such as serum, plasma, or urine). In this paper, we will demonstrate how a well-designed SPE sorbent (Oasis® HLB and Oasis® MCX) and straightforward SPE methods can accelerate and simplify the sample preparation. The Oasis® sorbent is a polymer-based sorbent; there are no silanol groups to complicate the mode of retention or method development. This novel sorbent, which is water-wettable and stable over the entire pH range, makes method development simple and fast.
With a generic 1-D reversed-phase SPE method, we are able to obtain very good results for a wide range of compounds including parent drugs and their metabolites, whether they are acidic, neutral, or basic. This generic SPE method is a very good starting point, and it is suitable for fast throughput analysis with LC/MS/MS detection. With a more selective 2-D reversed-phase SPE method, we are able to simultaneously isolate the analytes of interest from complex sample matrices and to eliminate almost all the interferences.
With a generic mixed-mode SPE method, we are able to selectively isolate the basic analytes from a complex sample matrix. Additionally, the same protocol is suitable for fractionation: acidic analytes and neutral analytes into one fraction, while the basic analytes into another fraction.
This presentation will cover the above three methods development strategies in great detail and gives numerous application examples from biological matrices (including urine, plasma, and serum). High and consistent results (>85% recovery with <5% RSD) are obtained for a wide range of analytes. Fast throughput analysis and high sensitivity detection with LC/MS (single-ion monitor and multiple-ion monitor) will be demonstrated as well.

 Authors: B. Alden, C. Gendreau, P. Iraneta, T. Walter

Affiliation: Waters Corporation

ABSTRACT

An organic/inorganic hybrid porous particle has been synthesized for use as an HPLC packing. This new chromatographic hybrid particle has been additionally surface bonded to attach multiple C. and C18 groups. These surface bonded hybrid particles not only exhibit extended pH stability, but show improved performance for basic compounds. On silica-based reversed-phase columns, broad and tailing peaks for many basic analytes have been attributed to surface silanols. We attribute the improved performance of the surface bonded hybrid particles to their reduced surface silanol concentration. Peak shape for a variety of basic, neutral, and acidic analytes was studied on reversed-phase bonded hybrid packings. Data is presented illustrating peak shape trends as a function of injected mass, mobile phase pH, and organic modifier.

 Authors: B. Alden, J. Carmody, J. Grassi, C. Gendreau, P.Irancta, T. Walter

We have recently developed a new family of reversed-phase HPLC packings based on organic/inorganic hybrid particles. To evaluate the hydrolytic stability of these packings relative to stateof-the-art C,,-silicas, we have used several different test protocols. These protocols include chromatographic tests using mobile phases containing buffers that range from pH 1.2 to 1 1 .5, as well as temperatures up to 60 oC. The results show that the packings based on hybrid particles exhibit exceptional stability in alkaline mobile phases and excellent stability in acidic mobile phases.

"HPLC Separations of Polar Compounds Under Highly AqueousConditions"

Author: Jeff Layne

Affiliation: Phenomenex, Inc

ABSTRACT

Despite the overwhelming popularity of classical reversed-phase HPLC columns, there are many separations that can be problematic under typical RP-HPLC running conditions. In particular, the analysis of highly polar analytes, such as those commonly encountered in the pharmaceutical and chemical industries, is complicated due to their poor retention on standard RP columns. Using high aqueous mobile phases (greater than 95%) can enhance retention of these polar analytes, but standard alkyl-bonded phases are susceptible to phase collapse under such conditions. In this seminar, we will discuss some alternative stationary phases that have been developed for use under highly aqueous conditions, and demonstrate how they can be applied to aid in the separation of highly polar compounds.

"IMPROVING PEAK SHAPE AND RETENTION OF AMINES
WITH A POLAR-EMBEDDED ALKYL PHASE"

Authors: J.W. HENDERSON, R.D. RICKER, and B.A. BIDLINGMEYER

Affiliation: Hewlett-Packard Company

ABSTRACT

A novel polar-embedded alkyl stationary phase bonded to ultra-pure silica offers exceptional liquid chromatography of polar analytes. Basic compounds can be difficult to analyze by alkyl bonded reversed-phase columns. At low pH, peak shape of basic compounds is improved by suppressing ionization of silanols. However in many instances, intermediate pH is necessary to improve selectivity, solubility, or stabilize a compound that is unstable at low pH. Intermediate pH may also negatively impact peak shape, by imparting a negative charge on silica surface. This polar-embedded alkyl stationary phase provides almost ideal peak shape over a wide pH range for amines. It also exhibits excellent performance in highly aqueous mobile-phase. Mild hydrophobic interactions render different selectivity than conventional C8 and C18 columns. These benefits give scientists one more valuable tool to ensure development of rugged methods for pharmaceuticals, especially amines.

"Capillary Electrophoresis for the Assessment of Non-Invasive
Sampling Techniques"

Authors: Valerie A. Frerichs, E. Peter Maziarz and Luis A. Colón

Affiliation: University at Buffalo, Departments of Chemistry

ABSTRACT

Using noninvasive methods allows for the assessment of a person's health in a painless, low-risk, and convenient manner.  Such
methods possess high patient compliance when used for monitoring in an at-home or surgical setting.  However, sampling substances non-invasively in a simple manner, while reliably reflecting the content of substances in blood is not a trivial task.    A suitable sampling technique must be thoroughly evaluated in terms of its feasibility for providing such characteristics before clinical studies can be initiated.   To this end, we are using capillary electrophoretic techniques to characterize and optimize two
sampling procedures, iontophoresis and passive transdermal diffusion.  We have chosen glucose as our model analyte to be
sampled, because of its biological importance.  Our studies have progressed to the point where sampling is executed in-vivo, such the we can make initial comparisons to blood concentrations.  In addition, we are now characterizing our samples for other analytes while optimizing our sampling procedures.  Here we present the most recent developments of our investigations.

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