Contributions to reversed-phase column selectivity: III. Column hydrogen-bond basicity.

Column selectivity in reversed-phase chromatography (RPC) can be described in terms of the hydrophobic-subtraction model, which recognizes five solute-column interactions that together determine solute retention and column selectivity: hydrophobic, steric, hydrogen bonding of an acceptor solute (i.e., a hydrogen-bond base) by a stationary-phase donor group (i.e., a silanol), hydrogen bonding of a donor solute (e.g., a carboxylic acid) by a stationary-phase acceptor group, and ionic. Of these five interactions, hydrogen bonding between donor solutes (acids) and stationary-phase acceptor groups is the least well understood; the present study aims at resolving this uncertainty, so far as possible. Previous work suggests that there are three distinct stationary-phase sites for hydrogen-bond interaction with carboxylic acids, which we will refer to as column basicity I, II, and III. All RPC columns exhibit a selective retention of carboxylic acids (column basicity I) in varying degree. This now appears to involve an interaction of the solute with a pair of vicinal silanols in the stationary phase. For some type-A columns, an additional basic site (column basicity II) is similar to that for column basicity I in primarily affecting the retention of carboxylic acids. The latter site appears to be associated with metal contamination of the silica. Finally, for embedded-polar-group (EPG) columns, the polar group can serve as a proton acceptor (column basicity III) for acids, phenols, and other donor solutes.

Wider pore superficially porous particles for peptide separations by HPLC.

Fused-core superficially porous particles have recently created considerable interest for high-performance liquid chromatography separations because of their unusual high column efficiency and much lower back pressure when compared to sub-2-microm particles. With superficially porous particles, larger solutes can move rapidly in and out of a thin porous shell, resulting in reduced band broadening at higher mobile phase velocities for greater separation speeds. The original silica fused-core particles were 2.7 microm in diameter with a 0.5-microm thick shell of 90 A pores designed for the fast separation of small molecules with molecular weights of less than approximately 5000. This manuscript describes new fused-core particles with similar physical characteristics except with a porous shell of 160 A pores designed specifically for rapidly separating peptides (and some small proteins) with molecular weights up to approximately 15,000 Daltons. Because of the larger pore size, restricted diffusion of these larger molecules is not seen since ready access to the entire porous shell is featured. Data are given to define sample loading qualities for columns of these new particles. Column stability studies indicate that these particles bonded with a sterically protected C(18) stationary phase can be used at low pH and higher temperatures with excellent results. The wider-pore particles of this study are shown to be particularly useful with a mass spectrometer detector for the rapid gradient separation of peptides using both volatile trifluoroacetic acid and formic acid containing mobile phases. Examples are provided for the separation of complex peptide mixtures to illustrate the capabilities for columns of these new wider-pore, fused-core particles.

Characteristics of superficially-porous silica particles for fast HPLC: some performance comparisons with sub-2-microm particles.

Columns of 2.7-microm fused-core (superficially porous) Type B silica particles allow very fast separations of small molecules at pressures available in most high-performance liquid chromatography instruments. These highly-purified particles with 1.7-microm solid silica cores and 0.5-microm-thick shells of 9 nm pores exhibit efficiencies that rival those of totally porous sub-2-microm particles but at one-half to one-third of the column back pressure. This presentation describes other operating features of fused-core particle columns, including sample loading characteristics and packed bed stability. The superior mass transfer (kinetic) properties of the fused-core particles result in much-improved separation efficiency at higher mobile phase velocities, especially for > 600 molecular weight solutes.

The art and science of forming packed analytical high-performance liquid chromatography columns.

Columns of packed particles still are the most popular devices for high-performance liquid chromatography (HPLC) separations because of their great utility, excellent performance and wide variety. However, the forming of packed beds for efficient, stable columns traditionally has been an art where the basics of how to form optimum beds generally was not well understood. The recent development of monolith rods was introduced in part to overcome the difficulty of producing stable beds of packing particles. However, these materials are less versatile than packed particle columns. Technology developments in recent years have produced a better understanding among those skilled in the practice of how to form optimized packed beds, and this has led to widely available, high-quality commercial columns. This presentation discusses the developments that led to the present state of column packing technology. Important steps in the packing of efficient, stable beds are described. The key step of selecting the best solvent for the slurry packing method is emphasized. Factors affecting the mechanical stability of packed columns also are discussed. The early art of packing columns now has evolved into a more scientific approach that allows the packing of good columns with a minimum of effort and time.

Development of some stationary phases for reversed-phase high-performance liquid chromatography.

The availability of a variety of stable organic stationary phases for columns has been a key factor in the development of HPLC as a major scientific tool. This paper explores the history and rationale used in the development of some important stationary phases and attempts to identify some of the strengths and limitations of these materials. Some of the author's experiences in stationary phase development illustrate approaches leading to present-day columns that exhibit a broad range of selectivity coupled with a high degree of reproducibility. Suggestions also are made for additional stationary phases that may be needed to complete column selectivity potential for HPLC separations.

Atypical silica-based column packings for high-performance liquid chromatography.

Column packings widely used for high-performance liquid chromatography (HPLC) mostly are based on porous silica microspheres with certain pore sizes and pore size distributions. Such materials have the most desirable compromise of properties that provide for effective and reproducible separations over a wide range of operating conditions. To provide desired separation characteristics, several manufacturers specially synthesize the silica particles for these packings. While such column packing materials have general utility for a wide range of needs, special silica-based particles have been synthesized with different physical conformations for special separation goals. This presentation describes some atypical types of silica-based particles with unique separation properties that enlarge the capabilities of HPLC methods.

Superficially porous silica microspheres for fast high-performance liquid chromatography of macromolecules.

Very fast reversed-phase separations of biomacromolecules are performed using columns made with superficially porous silica microsphere column packings ("Poroshell"). These column packings consist of ultra-pure "biofriendly' silica microspheres composed of solid cores and thin outer shells with uniform pores. The excellent kinetic properties of these new column packings allow stable, high-resolution gradient chromatography of polypeptides, proteins, nucleic acids, DNA fragments, etc. in a fraction of the time required for conventional separations. Contrasted with <2-microm non-porous particles, Poroshell packings can be used optimally with existing equipment and greater sample loading capacities, while retaining kinetic (and separation speed) advantages over conventional totally porous particles.

Functionalized porous silica microspheres as scavengers in parallel synthesis.

The use of solid scavengers in parallel solution-phase organic synthesis is an effective method for work-up and purification. Functionalized macroreticular or gel-form polystyrene particles are generally used for scavenging applications, how ever these materials have some limitations. We have developed new scavenging reagents based on ultrapure silica microspheres displaying a variety of functional groups useful for sequestering impurities from reaction products. These materials are easy to handle, have excellent mass-transfer properties, and are efficient scavengers in both polar and nonpolar organic solvents. The properties of these materials were tailored specifically to fit the needs of a medicinal chemist employing parallel synthesis techniques in current commercial equipment. Results are presented from head-to-head comparisons with conventional scavengers in tests designed to demonstrate the versatility of these new materials.

Ultrafast reversed-phase high-performance liquid chromatographic separations: an overview.

Ultrafast reversed-phase high-performance liquid chromatographic (HPLC) separations are often needed for analyses related to combinatorial chemistry, studies in liquid chromatography-mass spectrometry, and other applications in which very rapid sample turnaround is paramount. Unfortunately, no consensus exists regarding the best column technology for optimally performing the desired rapid separations. This overview compares the advantages and limitations for columns of ultramicroporous, ultramicrononporous, and superficially porous particles and monolith structures for the very fast separation of solutes by reversed-phase HPLC. Data from literature and the author's laboratory are used to illustrate the strengths and limitations of the various approaches that can be used for ultrafast separations.

Stability of silica-based, endcapped columns with pH 7 and 11 mobile phases for reversed-phase high-performance liquid chromatography.

The goal of this study was to define practical conditions and limitations of using silica-based, endcapped bonded-phase columns in intermediate and higher pH environments for developing rugged HPLC methods. Bonded-phase degradation in this pH range is a result mainly of silica support dissolution; covalently-bound silane ligands are hydrolyzed very slowly if at all from silica supports at intermediate and higher pH. Based on rates of silica support dissolution determined by chemical measurements and comparable chromatographic studies, we now find that endcapping alkyl-bonded stationary phases increases column longevity at pH 7, compared to non-endcapped columns. As previously determined for non-endcapped packings, we also find that the type of silica support determines the stability of bonded-phase packings. Silicas made by the sol-gel process are more resistant to dissolution than supports made by a silicate-gel (xerogel) process. In addition, endcapping methods apparently affect column stability, with double-endcapping methods apparently superior to single-endcapping approaches. Degradation rates for several endcapped commercial bonded-phase C8 columns were found to be quite variable in highly aggressive pH 7 accelerated-lifetime tests. Column stability in the pH 7-11 range is enhanced by using buffers other than phosphate in the mobile phase, and by excluding higher column temperatures. Certain silica-based endcapped bonded-phase columns can be used for developing rugged methods to at least pH 11 when used with organic buffers at < or = 40 degrees C.

Temperature as a variable in reversed-phase high-performance liquid chromatographic separations of peptide and protein samples. I. Optimizing the separation of a growth hormone tryptic digest.

Peptide and protein samples are often complex mixtures that contain a number of individual compounds. The initial HPLC separation of such samples typically results in the poor resolution of one or more band pairs. Various means have been suggested for varying separation selectivity so as to minimize this problem. In this study of a tryptic digest of recombinant human growth hormone, the simultaneous variation of temperature and gradient steepness was found to be a convenient and effective means of varying selectivity and optimizing the separation. The use of computer simulation greatly facilitated this investigation.

Temperature as a variable in reversed-phase high-performance liquid chromatographic separations of peptide and protein samples. II. Selectivity effects observed in the separation of several peptide and protein mixtures.

Changes in band spacing as a function of temperature and/or gradient steepness were investigated for four peptide or protein samples. Reversed-phase HPLC in a gradient mode was used to separate tryptic digests of tissue plasminogen activator and calmodulin. Additionally, a synthetic peptide mixture and a storage protein sample from wheat were studied. Simultaneous changes in gradient steepness and temperature were found to provide considerable control over band spacing and sample resolution. The effects of temperature and gradient steepness on selectivity in these systems appear to be complementary. Simultaneous optimization of both temperature and gradient steepness thus represents a powerful and convenient means of controlling band spacing and separation. Because of the complexity of these sample chromatograms, computer simulation proved to be a useful tool in both interpreting these experiments and in optimizing final separations.

Rapid, high-resolution HPLC separation of peptides using small particles at elevated temperatures.

Practical advantages are described for operating small-particle columns at elevated temperatures to increase the peak capacity, resolution or speed for separating peptides. Columns of small, highly-purified porous silica microspheres with a dense covalently bonded, bulky alkylsilane stationary phase permit continuous operation at temperatures of at least 90 degrees C. Operation at elevated temperatures decreases mobile phase viscosity and enhances solute diffusion, resulting in increased column plate number and separation resolution. Higher temperatures also decrease column back pressure, permitting longer columns of small particles for separating complex mixtures requiring large plate numbers. Use of a sterically protecting di-isobutyl-n-octadecylsilane stationary phase ensures stable and reproducible columns for operation at high temperatures, with the aggressive low pH mobile phase preferred for separating peptides. The monomeric nature of this phase ensures rapid mass transfer and high column efficiency.

Comparison of silica-based cyanopropyl and octyl reversed-phase packings for the separation of peptides and proteins.

The performance of a silica-based C8 packing was compared with that of a less hydrophobic, silica-based cyanopropyl (CN) packing during their application to reversed-phase high-performance liquid chromatography (linear trifluoroacetic acid-water to trifluoroacetic acid-acetonitrile gradients) of peptides and proteins. It was found that: (1) the CN column showed excellent selectivity for peptides which varied widely in hydrophobicity and peptide chain length; (2) peptides which could not be resolved easily on the C8 column were widely separated on the CN column; (3) certain mixtures of peptides and small organic molecules which could not be resolved on the C8 column were completely separated on the CN column; (4) impurities arising from solid-phase peptide synthesis were resolved by a wide margin on the CN column, unlike on the C8 column, where these compounds were eluted very close to the peptide product of interest: and (5) specific protein mixtures exhibited superior resolution and peak shape on the CN column compared with the C8 column. The results clearly demonstrate the effectiveness of employing stationary phases of different selectivities (as opposed to the more common optimization protocol of manipulating the mobile phase) for specific peptide and protein applications, an approach underestimated in the past.

Effect of interleukin-1 on the size distribution of cartilage proteoglycans as determined by sedimentation field flow fractionation.

The effect of interleukin-1 (IL-1) on the size distribution of cartilage proteoglycans was studied using sedimentation field flow fractionation (SdFFF), a rapid, high-resolution technique for the separation of proteoglycan monomers and aggregates. During incubation of cartilage in control media, 35S-prelabeled proteoglycan was lost primarily from proteoglycan present in the monomer form; aggregates were conserved. In the presence of IL-1, both 35S-proteoglycan monomers and aggregates were lost, suggesting that IL-1 increases the susceptibility of aggregates to loss from the cartilage matrix. Evaluation of uronic acid as a measure of net change in proteoglycan content indicated that IL-1 causes a net decrease in both monomers and aggregates. Kinetic studies suggested that aggregates are degraded to monomers which then diffuse out of the matrix. Incorporation of [35S]sulfate into cartilage proteoglycans following exposure to IL-1 showed that synthesis of monomers and aggregates is inhibited similarly. SdFFF is a valuable technique for studying proteoglycan metabolism. With its use, changes in proteoglycan monomer and aggregate populations can be detected in response to cytokines such as IL-1.

Growth of Staphylococcus aureus and synthesis of toxic shock syndrome toxin 1 in different in vitro systems.

Various complex and synthetic bacterial growth media have been used to study the growth of Staphylococcus aureus and the production of toxic shock syndrome toxin 1 (TSST-1) under certain in vitro culture conditions. Because of the biochemical and nutritional differences between these media and human menses, a program was designed to determine the growth and metabolism of S. aureus under conditions that more closely approximate in vivo conditions. Human menses, an artificial menses developed to match human menses, whole human blood, and complex bacterial culture media (with and without added whole human blood) were tested for the ability to support the growth of S. aureus and the production of TSST-1 in vitro. In addition, the impact of other organisms, commonly isolated from the human vagina, on the growth of S. aureus and the production of TSST-1 was evaluated. Results show that the environmental conditions provided by human menses are more closely approximated by the artificial menses and that neither commercial bacterial growth media alone nor complex media plus 25% or 50% human blood provide a menses-like environment for the growth of S. aureus and the production of TSST-1. Furthermore, the addition of a second organism to the S. aureus culture resulted in significant suppression of TSST-1 production, even when in vitro conditions were optimized for the production of TSST-1.

Response of various animal species to experimental infection with different strains of Staphylococcus aureus.

An experimental infection program was conducted in rabbits, pigs, and baboons with toxic shock syndrome (TSS)-associated and non-TSS-associated strains of Staphylococcus aureus to produce an animal model for TSS. TSS-associated strains of S. aureus--whether positive or negative for TSS toxin 1 (TSST-1)--could not be distinguished from non-TSS-associated strains of S. aureus by means of the rabbit whiffle-ball infection model; therefore, limited pilot infection studies were conducted in pigs and baboons. Experimental conditions were optimized in both the pig and the baboon studies to maximize the chance of producing TSS. Pigs infected with TSS-associated S. aureus strain CDC-11 developed some of the clinical signs observed in TSS (fever, hypotension, diarrhea, and vomiting). However, no changes were detected in clinical chemistry or hematology. Baboons infected with S. aureus strain CDC-11 showed only minimal signs of illness, i.e., lethargy, decreased food intake, and loose stools. TSS was not produced in pigs or baboons, even under optimal exposure conditions.

Effect of column degradation on the reversed-phase high-performance liquid chromatographic separation of peptides and proteins.

Many reversed-phase separations of proteins and peptides are currently performed in acidic mobile phases, e.g., 0.1% trifluoroacteic acid in water (pH 2) with organic modifiers. Such conditions are known to promote the cleavage of the silane from the silica in bonded-phase columns, especially for monomeric stationary phases. The stability of some columns commonly used for proteins and peptides has been examined, and it has been shown by both chromatographic and elemental analysis that degradation occurs very rapidly with fresh, "totally covered" column materials. Despite the loss of over half of the bonded phase in some cases, certain columns still exhibit adequate chromatographic performance, although reproducibility can be affected. The implications of these results with respect to both bonded-phase synthesis and mechanistic interpretation of chromatographic data is discussed.

Hormonal influence on experimental infections by a toxic shock strain of Staphylococcus aureus.

Subcutaneous infection chambers in rabbits were infected with a strain of Staphylococcus aureus isolated from a patient with toxic shock syndrome. Estrogens (mestranol and 17-beta-estradiol) protected male rabbits and prolonged survival. Neither androgens (testosterone and dihydrotestosterone) nor progesterone affected the susceptibility of intact or ovarihysterectomized female rabbits.