Microcapillary sampling

Acetylcholineesterase and choline oxidase A glassy C electrode surface was modified with osmium poly (vinyl-pyridine) redox polymer containing horseradish peroxidase (Os-gel-HRP) and then coated with a co-immobilized layer of AChE and ChO. A 22 pL pre-reactor, in which ChO and catalase were immobilized on beads in series, was used to remove choline. The variation in extracellular concentration of ACh released from rat hippocampal tissue culture by electrical stimulation was observed continuously with the online biosensor combined with a microcapillary sampling probe. Measurement of ACh and Ch was carried out by using a split disc C film dual electrode. 

Here functions Qnt X), Qj(X), and QP(X) can be determined experimentally using calibration samples. If these functions are linear independent then the parameters Ank, A, and Ap can be uniquely determined from the variation of P /1, , n2,. .. /( . /. / considered as a function of X. In particular, the side effects, i.e., the temperature and pressure dependences, can be eliminated from the transmission spectrum. The sensing method based on this simple idea was applied in Ref. 69 for determination of microfluidic refractive index changes in two microcapillaries coupled to a single MNF illustrated in Fig. 13.26c. The developed approach allowed to compensate the side temperature and pressure variation effects. 

Single-molecule detection in confocal spectroscopy is characterized by an excellent signal-to-noise ratio, but the detection efficiency is in general very low because the excitation volume is very small with respect to the whole sample volume, and most molecules do not pass through the excitation volume. Moreover, the same molecule may re-enter this volume several times, which complicates data interpretation. Better detection efficiencies can be obtained by using microcapillaries and micro structures to force the molecules to enter the excitation volume. A nice example of the application of single-molecule detection with confocal microscopy is... 

Matrices of monoisopropyl ester of PVM-MA were carefully applied in the lower conjunctival sac of rabbits. Inserts did not cause any irritation in rabbit eyes. Plasma and tear fluid samples were collected at different times during a period of 8 h. Blood samples were taken from the cannulated ear artery. Plasma was separated by centrifuging (2000 g, 4min) and kept at -20°C until analysed. Tear fluid samples (1 pi) were collected with microcapillaries at 30, 120, 240 and 480 min after application of the matrices. Tear fluid samples were diluted in 5 ml of phosphate buffer. 

Figure 3.2 outlines the application procedure. The sample to be analyzed is usually dissolved in a volatile solvent. A very small drop of solution is spotted onto the plate with a disposable microcapillary pipet and allowed... 

M. Galloway and S.A. Soper, Contact conductivity detection of polymerase chain reaction products analyzed by reverse-phase ion pair microcapillary electrochromatography, Electrophoresis, 23 (2002) 3760-3768. M. Masar, M. Dankova, E. Olvecka, A. Stachurova, D. Kaniansky and B. Stanislawski, Determination of free sulfite in wine by zone electrophoresis with isotachophoresis sample pretreatment on a column-coupling chip, J. Chromatogr. A, 1026 (2004) 31-39. 

Break several 5- or 10-//1 glass microcapillaries in half. Draw each sample into a microcapillary so that approximately equal volumes of air are present at either end. The microcapillaries can be cleanly broken by scoring them first with a diamond-tipped pencil or similar instrument. We use glass capillaries marked at the 5- or 10-//1 point by the manufacturer (VWR, San Francisco, CA). 

The application of CE for preparative separations of peptides and proteins is limited due to the low preparative capacity of capillary columns. In addition, adaptation of an analytical capillary system to a preparative one is not straightforward and requires certain modifications of CE instruments [2,22], Several procedures for fraction collection from a capillary have been developed recently, as reviewed in Ref. 22. For continuous fraction collection in CE it is necessary to modify the capillary outlet to complete the electrical circuit. Karger and coworkers achieved this by using a coaxial sheath liquid interface to transport the sample components leaving the capillary exit into the collection microcapillary... 

Microcapillary (0.200 -s- 0.300 mm diameter) and nanocapiUary (0.075 -t- 0.100 mm diameter) columns limit solvent consumption and interface more easily with mass spectrometer detectors. They can assay only small amounts of sample and are superior for managing Joule heating due to their enhanced surface area to volume ratio and lower volumetric flow rate (uL/min) for a given linear mobile phase velocity (mm/sec). 

LC ESI (8) mass spectra were recorded on a Finnigan TSQ700 instrument (San Jose, CA) in positive ion mode. A sample aliquot was injected into a fused silica microcapillary column with an inside diameter of 100 pm. The microcapillary was filled at the end with 10 cm of a C-18 reversed phase resin. PMP labeled oligosaccharides were eluted at ca. 1 pL/min directly into the electrospray ionization source with a 10 min gradient of acetic acid in H2O (0.5 %, v/v) to 80 % acetonitrile. Determination of experimental molecular weights was done with the deconvolution software provided by the manufacturer. 

Analyze your product by thin-layer chromatography. Dissolve very small samples of pure ferrocene, the crude reaction mixture, and recrystallized acetylferrocene, each in a few drops of toluene spot the three solutions with microcapillaries on silica gel plates and develop the chromatogram with 30 1 toluene-absolute ethanol. Visualize the spots under a uv lamp if the silica gel has a fluorescent indicator or by adsorption of iodine vapor. Do you detect unreacted ferrocene in the reaction mixture and/or a spot that might be attributed to diacetylferrocene ... 

The microcapillary packed and nano-LC colurtms are made of 0 0.5-0 5-mm-lD fused-silica tubes. The packing geometry of these colttrtms differs from that of a larger bore colunm, resulting in relatively higher colunm efficiencies. These type of colunms are frequently used in LC-MS applications with sample limitatiorrs, e.g., in the characterization of proteins isolated from biological systems. 

From a practical point of view, the discussion on flow-rate can be summarized as follows. In LC-APCI-MS, the typical flow-rate is 0.5-1.0 ml/min. For routine applications of LC-ESI-MS in many fields, extreme column miniaturization comes with great difficulties in sample handling and instrument operation. In these applications, LC-MS is best performed with a 2-mm-ID column, providing an optimum flow-rate of 200 pFmin, or alternatively with conventional 3-4.6-mm-ID columns in combination with a moderate split. In sample limited cases, further reduction of the column inner diameter must be considered. Packed microcapillary and nano-LC columns with micro-ESI and nano-ESI are rontinely applied inproteomics stndies (Ch. 17.5.2). 

The column inner diameter is determined by the amount of sample available and the LC-MS interface selected, tn general, flow-rates between 200 and 400 pl/min are considered optimum for (pneumatically-assisted) ESt. This explains the frequent use of 2-mm-ID columns, tn sample-limited analysis, e.g., in the analysis of mouse plasma samples, microbore (1 mm ID) or packed-microcapillary columns (320 pm ID) are applied at relatively low flow-rates [12-13]. For APCt, 4.6-mm-tD columns are preferred, operated at typically 1 ml/min. The LC system should provide symmetric peaks with a width that enables the acquisition of tO-20 data points for each compound in order to enable an accurate determination of the peak area. 

The volume that can be injected onto a packed microcapillary or nano-LC column is very limited, e.g., less than 0.1 pi for a 100-pm-lD column. This seriously compromises the achievable concentration detection limits, unless on-column preconcentration would be performed. However, for dilute sample solution, the injection volume is restricted by external peak broadening, and not by column loadability (typically -50-200 pg/g of porous packing material). Therefore, on-line SPE can be applied for sample preconcentration. 

The group of Hunt [1] pioneered the identification of trace-level peptides in complex biological samples by means of microcapillary reversed-phase RPLC-MS-MS in the analysis of endogenous human class I major histocompatibility complex (MHC) encoded human leucocyte antigen (HLA-A2.1) peptides. 

The bottom-up approach very much resembles classical protein identification strategies. The proteins in the proteome are first separated by 2D-GE (Ch. 17.3), or in some cases by SCX, size-exclusion (SEC), or affinity (AfC) chromatography. Specific proteins are excised from the gel, blotted, or electroeluted. The protein is digested, and the digest is analysed by LC-MS. The EC separation involves either RPLC with microcapillary or nano-LC columns (Ch. 17.5.2), or 2D-LC with typically SEC or SCX in the first dimension and RPLC in the second (Ch. 17.5.4). Alternatively, the sample may be introduced via either direct-infusion nano-ESl (Ch. 17.2), CE-MS (Ch. 17.5.6), or a microfluidic device coupled to MS (Ch. 17.5.5). 

The universal TLC facilities are utilized plates, adsorbents, microcapillaries, or micropipettes for sample application, development tanks, detection spray reagents, devices for spraying, and densitometers for quantification. Plates are either commercially precoated or handmade. Silica gel G (G, for gypsum as a binding substance), silica gel H (no binding substance) and, rarely, alumina and kieselguhr, form the thin-layer stationary phases. Complete sets of devices necessary for the preparation of handmade plates are commercially available. After the silica gel slurry is spread on the plates, they are left to dry in the air for at least 24 hr and shortly in an oven at 110°C. The plates are then ready for either direct use or for modification of the layer. From the great variety of precoated plates, which are commercially available and preferred nowadays, silica gel plates and plates with layers... 

Manual sample application employs various microcapillary pipettes and microsyringes. The microcapillary is one of the simplest and most useful methods for application of small sample volumes onto thin-layer plates. The capillary has a fixed volume of 0.5, 1, 2, or 5 pL, and accuracy is often better than 1%. The capillaries are supplied by the manufacturer in color-coded vials containing 100 pieces. The capillaries are handheld and can be positioned with a multipurpose spotting guide. 

Figure 4 Inhibition of ice recrystallization. Samples in 10 pi microcapillaries (740 pm diameter) were frozen and placed at -6°C and examined between crossed polarizing filters. Images were taken prior to, and after overnight incubation, but only the latter images are shown. From left to right samples include sample buffer controls (I, 2), bovine serum albumin, a control protein diluted to 0.2 and 0.02 mg/ ml, respectively (3,4), serial dilutions of 0.2, 0.02 and 0.002 mg/ ml Type I fish antifreeze protein in buffer (5-7), Chryseobacterium sp. cultures (8, 9), and E. coli cultures (10, 11). Note that only the fish AFP and the Chryseobacterium sp. cidtures have crystals too small to be detected at this magnification and the overlying feathery pattern, typical of snap frozen samples, is apparent. Bacterial cultures were at 2 x 10 CFU/ ml. Lines indicate duplicate samples and arrows indicate samples that were diluted.

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