Reported is the adaptation of a manual polysaccharide assay applicable for glycoconjugate vaccines such as Prevenar to an automated liquid handling system (LHS) for improved performance. The anthrone assay is used for carbohydrate concentration determinations and was scaled to the microtiter plate format with appropriate mixing, dispensing, and measuring operations. Adaptation and development of the LHS platform was performed with both dextran polysaccharides of various sizes and pneumococcal serotype 6A polysaccharide (PnPs 6A). A standard plate configuration was programmed such that the LHS diluted both calibration standards and a test sample multiple times with six replicate preparations per dilution. This extent of replication minimized the effect of any single deviation or delivery error that might have occurred. Analysis of the dextran polymers ranging in size from 214 kDa to 3.755 MDa showed that regardless of polymer chain length the hydrolysis was complete, as evident by uniform concentration measurements. No plate positional absorbance bias was observed; of 12 plates analyzed to examine positional bias the largest deviation observed was 0.02% percent relative standard deviation (%RSD). The high purity dextran also afforded the opportunity to assess LHS accuracy; nine replicate analyses of dextran yielded a mean accuracy of 101% recovery. As for precision, a total of 22 unique analyses were performed on a single lot of PnPs 6A, and the resulting variability was 2.5% RSD. This work demonstrated the capability of a LHS to perform the anthrone assay consistently and a reduced assay cycle time for greater laboratory capacity.

The efficacy of the particle beam LC/FT-IR interface, in its development as a tool for the determination of dynamic protein structure from experiments such as HPLC separations and folding/refolding intermediate analysis, is presented here. The particle beam apparatus can be used to desolvate proteins rapidly in preparation of IR measurements. Several experiments have been designed to determine whether the operation of the particle beam apparatus causes alteration to the complex structural features of globular proteins, and whether it produces a solid-state spectrum representative of protein solution structure. It is shown here that the structural integrity of β-lactoglobulin is maintained when nebulized, desolvated from solution, and deposited onto the IR-substrate. Since enzyme activity is dependent upon the maintenance of higher-order structure, a complementary series of spectrophotometric-activity experiments with lysozyme collected from the particle beam were performed to determine the state of the tertiary and quaternary structures. The lysozyme particle beam deposit not only produced a secondary structure estimate similar to that of solution; it also retained its biological activity. It is demonstrated that the particle beam can induce structural changes in proteins with a carrier-liquid concentration gradient; this characteristic is useful for band assignment.

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The Particle Beam LC/FT-IR interface has been modified so that the aerosol produced can be electrostatically charged by the induction charging (IC) technique. 1,2 Manipulationof the beam of uniformly charged particles, by electrodynamics using potential arrays, in addition to physical alignment, enhances analyte transport efficiency. The emphasis of this work involves FT-IR analysis of biomolecules prepared from an electrostatic aerosol. With IC, and potential influence over the trajectories, collection of the desolvated biomolecule can be achieved without the high-energy impact associated with normal Particle Beam operation, an ideal situation for conformation studies in which structure is often affected by the harsh collision. In this work the globular proteins, a-Chymotrypsin, Papain, and the amino acid d, l-Tryptophan were electrostatically charged by the IC technique, and deposited onto IRtransparent substrates which produced films that were examined by FT-JR. INTRODUCTIONOur laboratory is presently involved in the use of the Particle Beam LC/FT-IR interface for the evaporation of water from biomolecules. The rapid desolvation of the aqueous medium from these compounds, while they are suspended in an aerosol, affords several FT-JR conformational studies otherwise difficult by solution techniques (e.g. denaturation, intermediates). Since no heat is involved, the Particle Beam can produce a gas-phase beam of biomolecules without any structural degradation. Upon completion of desolvation, the more volatile solvents are removed from the analytes by way of a momentum-based separator. Analytes then impact onto an JR-transparent substrate to produce a dried film that is later removed and examined by FT-IR spectrometry. At the present time the Particle Beam device has an efficiency of approximately 30 % (that is, 30 % of any solute introduced to the device is recovered upon analysis). Charges can be readily induced on nebulized liquids. The goal of this work is to produce a beam of charged biomolecules, which can be steered and controlled by electrodynamic means, so to improve transport efficiency. In addition, this capability can be used to decrease the energy of the analyte as it impacts onto the substrate; thus any conformational changes that might occur during collection can be avoided. We report here the development, and determination of the efficacy of induction charging, with biological molecules, as incorporated into the particle beam apparatus. Principles of Induction ChargingThe induction charging process produces a dense surface charge on the aerosol droplets produced by the Particle Beam. This is accomplished by passing the liquid jet through a cylindrical electrode placed coaxial with the beam. A potential is applied between the cylinder and the jet by connection to the liquid orifice (similar to a coaxial capacitor with the jet as the conductor). The longitudinally positioned jet breaks apart inside the electrode, and electrical charges are trapped upon the individual droplets. Since this aerosol produ...