fall 97 program

Abstract: HP will discuss Capillary Electrophoretic Chromatography, and share some insights into the current state of the technique. This discussion will include real life application as well as some current comparisons to other chromatography techniques.

Time 9:30 AM

Title: CE-LIF Analysis of Subcutaneous Fluid Sampled Noninvasively

Authors: Valerie A. Frerichs and Luis A. Colon

Affiliation: SUNY at Buffalo

Abstract: Research presented here aims to develop non-invasive sampling procedures for chemical/clinical analysis. Our current focus is the development of in-vivo sampling procedures for the extraction of substances from subcutaneous fluid. Methods of sampling employ iontophoresis and passive diffusion to bring small amount of fluid to the skin surface from the subcutaneous tissue. Such procedures are more comfortable and pose little risk to the patient. Samples are analyzed for glucose content. Glucose was chosen as the substance of interest because of the analyte's biological importance. A relative fast and simple means of analysis has been achieved using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF).

We will present the methods to non-invasively sample subcutaneous fluid, and to determine glucose using CE-LIF. The CE-LIF method offers detection limits in the nanomolar range. In addition, results for in-vivo samples using human subjects will be presented.

Time 10:50 AM

Topic Presentation of NERCDG National Chromatography Award

Time 11:00 AM

Topic Chromatography on Electrophoretic Platform: Capillary Electrochromatography

Author Featured Speaker - Csaba Horvath

Affiliation Yale University - Department of Chemical Engineering

Abstract Recent advances with fused silica capillaries in gas and liquid chromatography as well as in electrophoresis have paved the way to the emergence of capillary electrochromatography (CEC) that employs capillary columns packed with a suitable stationary phase and high electric field to generate electroendosmotic flow (EOF) of the eluent. In essence chromatographic separations are carried out in an apparatus similar to that for capillary zone electrophoresis and CEC is used for separating both neutral and charged species. Our main research interest is the control of the magnitude and steadiness of EOF, the enhancement of intraparticulate mass transfer and column stability under conditions imposed by the high electric field and to demonstrate the merits of CEC in various applications. Experimental results show that the magnitude of EOF is determined by the electrokinetic permeability of the bed, the applied voltage gradient, and eluent properties such as the ionic strength, viscosity etc. The electrokinetic permeability depends both on the hydraulic permeability of the packing and the charge density at the chromatographic surface. At present CEC mostly employs siliceous stationary phases that have at basic pH high negative charge density due to the abundance of dissociated silanol groups. Upon functionalization with appropriate ionic groups, however, polymeric supports were made with comparable charge densities in our laboratory and the columns thus obtained were characterized by their conductive and electrokinetic properties. Furthermore by in situ polymerization a fluid impervious thin tube is fabricated inside of the capillary to prevent contract between the silica innerwall of the capillary and the aqueous electrophoretic medium. The innerwall of "the tube in the tube" is functionalized to carry fixed negative of positives charges. The overall mass transfer is affianced in CEC with respect to that in traditional HPLC. The reason for this is twofold. EOF is generated at the surface of the packing, with concomitant reduction of interstitial band spreading. Further, in high electric field EOF is believed to occurred inside the pores of the stationary phase particles so that intraparticulate mass transfer resistance's would be relaxed by this "electroperfusion' effect. The two phenomena appear to be responsible for the enhancement of column efficiency in CEC with respect to that in RPLC. At high voltages the electric field is strongly non uniform in and around the stationary phase particles. As a result degassing of the eluent occurs at the hot spotsand bubbles are formed and (lie charged sorbent particles are readily delocalized by the high electric field to the detriment of column stability. In order to overcome this problem monolithic packed columns were prepared either by in situ polymerization of a rigid macroreticular rnatrix or by sintering the particles constituting the column packing our laboratory. Functionalization provided chromatographic surface and a stable packing without the need for irkome supporting frits at the column ends. On the basis of the experimental results so far, CEC is emerging as a separation technique of potentially high peak capacity. It is because relatively long columns packed with very small particles can be employed with concomitant enhancement of the plate efficiency.

Time: 1:30 PM

Title: Determination of EDTA in Urine and Plasma by CE/MS/MS

Authors: Robin L. Sheppard, Jack Henion

Affiliation: XEROX Corporation

Abstract: A quantitative method has been developed for the determination of EDTA in Human plasma and urine. The samples are prepared with automated anion-exchange solid-phase extraction using 100 microliters of human plasma. The extracts are analyzed by capillary electrophoresis/mass spectrometry using selected reaction monitoring (SRM) in the negative mode. Large volume injections (10% of CE capillary volume) are used to improve the concentration level of detection via field-amplified sample injection. The first reported method validation of a CE/MS/MS technique was carried out for this method. Using a 13C stable-label isotope as the internal standard, the lower level of detection and lower level of quantification were determined to be 7.3 ng/ML and 14.6 ng/mL EDTA in human plasma receptively. The injection precision had a relative standard deviation (RSD) of 6.1%. the intra-assay precision was less than 15% RSD. The intra-assay accuracy was less than +/- 12% bias from the nominal concentration. The inter-assay precision was less than 18% RSD and the inter-assay accuracy was less than +/- 9% bias from the nominal concentration.

Time: 1:50

Title: Evaluation of Columns Packed for Capillary LC and Capillary

Electrochromatography

Authors: Adam M. Fermier and Luis A. Colon

Affiliation: SUNY at Buffalo

Abstract: Capillary LC and capillary electrochromatography are advantageous due to the higher resolving powers. However, packing capillary columns is complicated by the size and length of the columns. Typically these columns are packed using pressure. An alternative approach that we have been investigating is the use of centripetal forces to pack the columns. Advantages of this approach include ability to pack multiple columns at once and the impact velocity is actually greater than pressure packing. A device was designed and constructed to allow the particles to pack radially around a central slurry reservoir. The device and packing procedure along with the chromatographic data supporting the alternative use of centripetal forces for packing liquid chromatographic columns will be presented.

Time: 2:10 PM

Title: Aroma Analysis as a Complement to Gas Chromatography

Authors: Jennifer A. Cyr

Affiliation: AromaScan Inc.

Abstract: Gas chromatography is used extensively for quality control in many industries including fine chemicals, personal care products and pharmaceuticals. Component analysis can be used to verify that a product meets a standard specification established by a manufacturer. There are, however, a few drawbacks to this analytical technique including involved sample preparation, length of analysis and level of training necessary. As a complement to GC, the Aroma-Scanner can be used as a screening tool to ensure product quality measurement though aroma is difficult to determine based on GC results when a product odor my be due to a chemical that is present a low PPM or pp. level. There is little to no sample prep needed, analysis times are generally under ten minutes and instruments can be run by any user from the research and development lab to the production floor. Off odors and non-compliance can be caught quickly. Once a rejected product is detected, further component analysis can be completed to determine the source of the contamination. If the product is acceptable it can be released for use in a matter of minutes.

Time: 2:30 PM

Title: ProSep 800 - A New Injection Technique

Authors: Larry Anderson

Affiliation: Environmental Sample Technology, Inc.

Abstract: The ProSepTM systems are designed to provide enhanced sensitivity and increased speed of analysis. These systems represent a vast improvement in current Gas Chromatographic technology. ProSep 800 TM extends the length of your injection port from 78 mm to 240 mm by adding a temperature control zone to the base of a Hewlett Packard split/splitless injection. The longer pre-column dimension greatly increases the resolving capabilities of the system allowing larger (1-100 microliter) and faster injection using current HP autosamplers. Different pre-column packing materials and wall coated open tubular configuration for the precolumns further optimize the system for specific applications. Partitioning materials optimize the system by decreasing breakdown and give the inlet the capability to selectively transfer mass to the analytical column or out the purge vent. High Temperature capability (800 degrees C) allow higher molecular weight compounds to be analyzed faster with less carryover.

Time: 3:40

Title: Solid-Phase Extraction Method Development Strategies for the

Determination of Drugs in Biological Matrices

Authors: Yung-Fong Cheng, Laura Bean, Ed Bouvier, Mark Capparella,

Pam Iraneta, Uwe D. Neue, and Dorthy J. Phillips

Affiliation: Waters Corporation

Abstract: Solid-Phase Extraction (SPE) has become a widely used sample preparation procedure for extraction of drugs from biological fluids, such as plasma, serum, or urine. The most commonly used sorbents are porous silica particles derivatized with C18 or C8. However, there are several disadvantages for silica-based reversed-phase SPE sorbents: undesirable silanol activity, pH limitations, breakthrough of polar analytes, irreproducible and unreliable recoveries, hydrophobic collapse and stopcock manipulations. These limitations have caused SPE the bottleneck in today's high productivity laboratories.

In this paper, we address the specific reasons for causing low and variable recovery on traditional reversed-phase SPE sorbents. We also demonstrate how a well-designed sorbent, OasisTM HLB extraction sorbent, and a well-thought out SPE method development strategy can simplify and accelerate the SPE process. Recoveries greater than 85% and RSDs less than 5% will be shown for a wide range of compounds (including acids, neutrals, and bases) in porcine serum and plasma matrices. This data will show the universality of the OasisTM sorbent which enables parent drugs and their metabolites to be analyzed simultaneously.

Time: 4:00

Title: HyPURITY TM Elite: The Next Generation C18

Authors: Cathy Burchat

Affiliation: Hypersil

Abstract: As new development are made in the fine chemical industry, the continued trend towards demanding application and automated chromatographic analysis has meant that the stability of the chromatographic support has become of key interest. In chromatographic terms, this has required the development of stationary phases, which can analyze a wide range of compound types with a single analysis on a reproducible basis, with good stability even at extremes of pH.

The performance of the C18 phases are primarily affected by the alkyl chain, but secondary interaction can be attributed to a variety of causes including hydrogen bonding, ion exchange, analyte-silica interaction, and interactions with residual silanol groups. These interactions can give rise to poor chromatographic performance in terms of peak shape as well as, poor reproducibility and chromatographic behavior. A series of chromatographic tests are employed on HyPURITY TM Elite which not only characterize the surface for specific interaction types, but also show how the tests may be used as a means of assessing batch-to-batch reproducibility. Several other popular and newer commercially available C18 phases are also investigated using these chromatographic tests, with comparisons shown. A few applications of HyPURITY TM Elite will also be presented.

Time: 4:20 PM

Title: High Speed HPLC Analysis and Purification for Combinatorial

Chemistry

Authors: Ernest Sobkow, Peter C. Rahn and J. Robert Bickler

Affiliation: YMC, Inc.

Abstract: Traditionally, the search for biologically active molecules has involved the synthesis of one compound at a time. New combinatorial chemistry techniques have reduced synthesis time by generating a large number of chemical variants at one time. The resultant combinatorial library is subjected to high throughput screening techniques. This paper shows how rapid, rugged HPLC separation with specially designed YMC, Inc. CombiChromTM columns provides quick turnaround time. Reducing analysis time from the traditional 30 minutes to less than 5 minutes allows chemists to analyze and isolate hundreds of compounds daily. Minimizing analysis time is paramount to meet the major challenge of isolating the plethora of compounds generated by the combinatorial chemistry technique. This paper shows how to quickly isolate the target compound from all the inactive combinations. These same separations are scaled up to larger diameter columns without sacrificing turnaround time.

Time: 4:40

Title: Electrochemistry: Amperometry in HPLC

Authors: William M. Pastore IV

Affiliation: Dionex Corporation

Abstract: Many compounds that are normally detected via UV Spectroscopy afford detection via Amperometry. The advantage of the use of amperometry is that this technique will allow the detection of non-chromophoric compounds as well as chromophoric compounds. There is little interference from the solvent in amperometry and the limits of detection are orders of magnitude lower than UV Spectroscopy.

NERGDG Fall 97 Posters

Poster Title

Remote Field Sampling Using Solid Phase Microextraction (SPME)

Isolating Impurities form Drugs and Vitamins with Bonded Normal Phase (BNP) Media

Carotenoid Analysis in Food and Drug Formulations

Controlling Selectivity of Polar Molecules on Reversed-Phase Columns

Alternative to Normal Phase Silica Separations for Rugged HPLC Separations

Recovery of Volatile PAHs as a Function of Novo-Clean C18 Membrane Drying Time

Fatty Acid Methyl Ester Analysis on a Thermally Stable, High Cyanopropyl Capillary GC Column

Separation of Nucleotides from Nucleosides and Bases with Novo-Clean Silica Teflon Extraction Membranes

Reducing Gas Chromatography Analysis Time with the New High Temperature Multicapillary Column

Comparison of Inertness Properties of Tubing in Gas Chromatography

Packed Capillaries and Specialty Phases for Capillary Electrochromatography

An N-Octyl Stationary Phase for Reversed-Phase HPLC Using Highly Aqueous Mobile Phases

Capillary Electrochromatography-Performance Evaluation

Analysis of Molecular Sulfur by HPLC/EC

Controlling Extra-Column Dispersion in Microbore and Capillary HPLC

Posters

Title: Remote Field Sampling Using Solid Phase Microextraction (SPME)

Authors: Raymon Midrup, Bober Shirey and Venkantachalam Mani

Affiliation: Supelco, Inc.

Abstract: Solid Phase Microextraction (SPME) has been used for the extraction of a broad range of analytes from a variety of matrices. With the development of new fibers incorporating porous carbon, the collection and retention of volatile analytes has been improved. The ability to extract the variety of pesticides, without polarity constraints, has also been demonstrated using the polysiloxane coated fibers. Using the extraction capability of both fibers in field sampling provides the analyst a simpler approach to environmental monitoring.

Data will be presented showing the ability of SPME to retain analytes after sampling. A field sampling SPME device that will enable technicians to extract samples in the field and bring the device back to the laboratory for testing will be discussed. Storage and recovery of both analyte classes will be shown to exceed three days.

Title: Isolating Impurities from Drugs and Vitamins with Bonded Normal-Phase

(BNP) Media

Authors: Peter C. Rahn and J. Robert Bickler

Affiliation: YMC, Inc.

Abstract: The degradation and metabolism route for many drugs results in the addition of polar functional groups to the parent molecule. This presentation compares preparative separation on bonded normal phase (BNP) media to bare silica media for isolating polar compounds. The preparative capacity for BNP media is compared to bare silica. The advantage of short columns to predict the preparative capacity for larger columns is also evaluated. A major benefit of BNP media for preparative separation is their insensitivity to water and other polar contaminants that are present in most samples. The BNP media is easily cleaned and regenerated with common polar solvents such as methanol and water. Even after washing with polar solvents, the BNP media retains their ability to separate polar impurities on subsequent runs.

Title: Carotenoid Analysis in Food and Drug Formulations

Authors: Peter C. Rahn and J. Robert Bickler

Affiliation: YMC, Inc.

Abstract: Interest in the perceived health benefits of carotenoids continues to require their separation and quantification. The minor shape difference among caroteniod isomers and the separation of individual species has been very challenging. Official carotenoid methods generally do not account for isomers that are biologically active. Published data on vitamin A content of foods is inaccurate since not all the isomers are measured. To illustrate this problem, various foods are analyzed for their carotenoid isomer content. Their carotenoid profiles for foods before and after exposure to high temperatures are shown. This study employs the YMC Inc. Carotenoid column for reversed phase HPLC separations that is highly selective for the carotenoid geometric isomers. The utility of this tailored phase is demonstrated for the separation of carotnoids in foods, dietary supplements, pre-formulation concentrates, and vitamin formulations.

Title: Controlling Selectivity of Polar Molecules on Reversed-Phase Columns

Authors: Peter C. Rahn and J. Robert Bickler

Affiliation: YMC, Inc.

Abstract: Polar and ionizable compounds are difficult to separate by reversed-phase chromatography without the use of complex buffers or ion paring reagents. In this study we show varying pH can be beneficial to control retention and selectivity of ionic compounds in reversed-phase chromatographic on ODS (C18) columns. Differences between a standard ODS phase with trimethylsilane end capping and a unique ODS phase with hydrophilic end capping (YMC-ODS-AQ) shows this new specialty phase is ideal for very polar molecules. In addition to pH, selectively achieved on this new phase is also controlled with ionic strength and buffer composition. The major effects on polar molecules are greatest when the organic solvent concentration is less than 40% of the mobile phase.

Title: Alternative to Normal-Phase Silica Separations for Rugged HPLC

Separations

Authors: Peter C. Rahn and J. Robert Bickler

Affiliation: YMC, Inc.

Abstract: The successful HPLC separation of compounds containing polar functional groups is difficult by reversed-phase columns. The selectivity and resolving power of normal-phase is higher that reversed -phase for separating polar isomer. Historically, normal-phase separations are shunned due to reproducibility and low recovery. Reproducibility problems with silica or alumina columns are caused by varying acidity levels, deactivation and hydration by polar solvents, polar samples and the presence of water. To eliminate reproducibility problems and still have good resolution for polar compounds, a new family of bonded phases is introduced. These new phases dramatically improve the reproducibility, ruggedness and mass recovery of normal-phase separations. Alcohol or water does not deactivate these new phases. In addition these new polar bonded phases provide a different selectivity and better peak symmetry for polar compounds compared to either bare silica or reversed-phase separations.

Title: Recovery of Volatile PAHs as a Function of Novo-Clean C18 Membrane

Drying Time

Authors: Gary L. Nixon, T. R. West and M. A Waksmonsky