Sample application syringe

Semiautomatic devices suited for preparative purposes are the CAMAG Linomat 5, the Desaga HPTLC applicator AS 30, and the Alltech TLC sample streaker. For all devices, the syringe has to be filled manually with sample solution and rinsed after sample application. Except for the Alltech TLC sample streaker, each of these instruments can be employed either as software-controlled or as a stand-alone device. The former is more convenient for creation, editing, and saving of the application pattern and instrument parameters. 

Figure 13.3. Sample application tools from top to bottom, glass capillary, Pasteur pipette with tip drawn out, syringe for HPLC, and syringe for GC. 

It is necessary to rinse the syringe very carefully with buffer (at least five times) and with the next sample between two sample applications. 

The Linomat-5 is the latest development in a long line of CAMAG sample applicators, using the spray-on technique in qualitative, quantitative, and preparative TLC/ HPTLC (Fig. 5). This applicator sprays samples, preferably in the form of bands of selectable length or as spots. The spray-on technique enables larger sample volumes to be applied to the layer by contact transfer. The sample dosage syringe is selectable, 100 or 500 pL. 

If you have to perform assays by quantitative TLC, you will find fully automatic sample application indispensable. This will at least require the Automatic TLC Sampler (ATS 3) of CAMAG (Fig. 34), in which a PC controls the application and then, in combination with a TLC scanner, the evaluation. The PC also processes the measured data. The samples are applied from a steel capillary, which is connected to a dosing syringe driven by a stepping motor. The samples are applied to precoated layers (on glass or foil), up to the 20 x 20 cm format, either spotwise or bandwise as required. Applica-... 

At lower pressure (<7-10.5 MPa) syringe injection of the sample through a membrane is possible, but mostly loop injection valves are used for sample application. These valves may be operated at a pressure up to 42-49 MPa. 

S.3.2 Syringe injection. This was the first type of sample application system developed and closely resembled GC and involved syringe injection of the sample through a self-sealing septum. The greatest column efficiency is achieved by application of the sample via a syringe directly onto the column bed, due to... 

On column. One on-column sample application method involves stop-flow techniques. In this instance, the pump is stopped and isolated by a three-way valve from the column, the sample is then loaded via a syringe through an injection port which does not contain a septum. The pump is then restarted, the flow restored by switching the valve, and the sample is rapidly flushed onto the column. There is no apparent loss in efficiency but inaccuracies in retention measurement occur due to the finite time required for flow to be established and therefore this technique is redundant. 

Several fully automated spray-on sample applicators are available. In one device, a motor driven syringe is used to suck up sample volumes of 0.1 to 50 p.1, which are then deposited as spots or bands on the layer [104]. The syringe feeds a stainless-steel capillary connected to a capillary atomizer. The applicator can be programmed to select samples from a rack of vials and deposit fixed volumes of the sample, at a controlled rate, to selected positions on the layer. The applicator automatically rinses itself between applications and can spot or band a whole plate with different samples and standards without operator intervention. A number of multi-sample applicators for the simultaneous transfer and deposition of several samples at the same time have been described [106-108]. 

Manual application of samples is performed by touching a capillary tube containing the sample to the plate or by use of a hypodermic syringe. A number of mechanical dispensers, which increase the precision and accuracy of sample application, arc now offered commercially. 

Deactivated florisil (S grams) was added to a disposable, polycarbonate, syringe barrel fitted with a glass-wool plug. The resin was then washed with 20 mL of hexane immediately prior to sample application. 

Fig. 2 Diagram of the twin-chamber injector with a needle in position of sample application. 1 - quartz sinter, 2 - steel core, 3 - magnet, 4 - quartz tube, 5, 6 - lower and upper housing, 7, 10, 12 - ball valve, 8 - injecting cylinder, 9 - solvent evaporating chamber, 11 - analyte evaporating chamber, 13, 14 -electric heater, 15, 16, 17 - radiator, 18, 25, 26, 32 - needle valve, 19, 27, 28, 31 - solenoid valve, 20, 23 - carrier gas inlet, 21 - chromatographic column port, 24 - six-way valve, 29, 30, 33 - auxiliary gas inlet, 34 - connector pipe for sample introduction, 35 - micro-syringe needle channel, 36 - membrane, 37 - auxiliary gas outlet.

Once the sorbent type has been selected, extraction of the sample should be confirmed. The cartridge should be conditioned with 1-2 bed volumes of a solvent such as methanol, then prepared for sample introduction by passage of 10-20 bed volumes of the sample solvent. If an ion-exchange phase is used, it should first be converted to the proper counterion, then equilibrated at the appropriate pH. Because of the slow diffusion rates of macromolecules, samples should be applied at relatively low flow rates, preferably less than lOmL/min a cartridge syringe device can generate flow rates of 200 mL/min [3]. After sample application, the bed should be washed twice with 10-20 bed volumes of sample solvent followed by analysis of both washes by HPLC. Absence of sample components in the chromatogram indicates adequate extraction by the sorbent, whereas appearance of samples in the wash implies that a more retentive sorbent is needed. 

Automated or semiautomated sample applicators are available as well. These devices apply consistent and reproducible sample spots, but it is a misconception that they are necessary for quantitative work. With proper technique, manual methods of sample application can provide results entirely comparable to those from automatic devices. Commercial automated units employ syringes or rows of microcapillaries to apply a spot or band of sample, and some actually .spray the sample onto the layer. Many are designed for preparative separations, applying large amounts of sample as streaks across the sorbent layer. 

Plate development may be described in terms of either time or distance. With timed development a plate is allowed to develop for a. specific period of time, after which it is carefully lifted out of the chamber. The location of the solvent front is then marked with a pencil or a sharp object such as a syringe needle. If mobile phase is instead expected to travel a fixed distance (e.g, 10 cm), this distance should be marked on the plate at the time of sample application. It then becomes easy to observe when the mobile phase has reached the desired point so the plate can be removed promptly from the chamber. A fixed development distance is used to standardize R values because... 

HPTLC uses much finer (about 5 micron) particles and somewhat thinner layers (200 micron) than TLC. This leads to much less spreading on the plate. Smaller plates are used (10 x 10 cm as opposed to 20 x 20 cm) and this necessitates better sample application of smaller samples. This is best carried out with an automatic spotter rather than with a micro-syringe, and with a steady hand and eye. The qualitative and quantitative... 

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