Degassing the Eluent

Degassing the eluent is important because air can get trapped in the check valve (discussed later in this section), causing the pump to lose its prime. Loss of prime results in erratic eluent flow or no flow at all. Sometimes only one pump head will lose its prime and the pressure will fluctuate in rhythm with the pump stroke. Another reason for removing dissolved air from the eluent is because air can result in changes... 

Sodium hydrogen carbonate, NaHCOj, 1.7 mM when used. 14.3 g/L as a 100 x concentrate 10 mL/S Helium, for degassing the eluent. 

The traditional HPLC instrument is composed of two different parts the first part separates the components of the sample and the other part accomplishes the detection of the components separated. The part of the HPLC carrying out the separation contains a column, an injection device and the eluent delivery system (pump with filters, degasser and transfer tubing, eventually a mixer for gradient elution). One or more detectors, a signal output device coupled with appropriate software, are responsible for detection and primary data evaluation. Pumps deliver the eluent or the different components of the eluent into the column with a precise, constant and reproducible flow rate. 

Degassing of the eluent is important for trouble-free operation and highly sensitive detection,2 otherwise the eluent may become supersaturated with air that is released as bubbles in the pump check valves or the detector flow... 

A simple and effective form of degassing is to hold a flask of mobile phase under a vacuum while agitating the contents in an ultrasonic bath (Fig. 3.7). The eluent is then transferred to the chromatograph for several hours of reliable operation, especially if the eluent is blanketed with an inert gas such as helium. This approach is particularly useful for clean solvents that readily absorb gases such as carbon dioxide from the atmosphere. The eluent should be degassed in the solvent storage container used for chromatography to minimize contact with the air. 

Procedure (See Chromatography, Appendix BA.) Use a suitable high-performance liquid chromatograph equipped with differential refractometer, autosampler injection unit, mobile-phase degasser, column heating block or oven, and a computing integrator. The column is Lichrosorb RP-18 250-mm x 4.5 mm (id) (GL Science, Inc., or equivalent) and YMC-Pack ODA-A A-303 250-mm x 4.5 mm (id) (YMC Company, Ltd., or equivalent) connected in a series, or equivalent, maintained at 50°. Use 80 20 acetone acetonitrile as the eluent, at a flow rate of 2 mL/min. 

The major operating problem of piston pumps is dissolved air and the formation of bubbles in the eluent. Bubbles in the pump heads cause pulsation of volume flow and pressure pulsation. Bubble formation and cavitation problems are promoted at the inlet check valve because the minimum pressure in the system is reached here. For this reason the eluent must be suitably degassed. This can be done online by a membrane degasser, by pearling helium offline through the eluent or by the use of an ultrasonic bath. 

To a 100-mL, two-necked flask, is added a mixture of cyclohexanone (5.9 g, 60 mmol), o-iodoaniline (4.4 g, 20 mmol), and 1,4-diazabicyclo[2.2.2]octane (DABCO) (6.7 g, 60 mmol) in N,N-dimethylformamide (DMF) (60 mL). The mixture is degassed three times via nitrogen/vacuum, followed by the addition of palladium acetate (Pd(OAc)2) (2.24 mg, 0.1 mmol) (Note 1). The mixture is degassed twice and heated at 105°C for 3 hr or until completion of the reaction (Note 2). The reaction mixture is cooled to room temperature and partitioned between isopropyl acetate (150 mL) and water (50 mL). The organic layer is separated, washed with brine (50 mL), and concentrated under vacuum to dryness. The residue is chromatographed on 50 g of silica gel using 700 mL of ethyl acetate-heptane (1 6) as the eluent to give 2.22 g of 1,2,3,4-tetrahydrocarbazole (65%) as a pale brown solid (Note 3). 

For various reasons the eluents used for HPLC should be degassed, especially the polar ones which can dissolve fairly high amounts of air (water, buffer solutions, all other aqueous mixtures, water-miscible organic solvents), otherwise some problems may occur gas bubbles will show up in the detector where the back pressure is low, the baseline will be noisy or spiked with extra peaks, analytical precision may be affected, and peaks may be small because dissolved oxygen absorbs at low UV wavelengths and quenches fluorescence. Gradient separations are particularly delicate as gas is released when air-containing solvents are mixed. 

Many of the solvents normal to HPLC use can be used for the eluent make-up. The additional conductivity required for electrochemical detection is provided by the addition of an electrolyte in the range of 1 to 10 g/L, yielding conductivities of 1 to 10 mS/cm. The eluent is degassed in the usual manner, using hehum or a vacuum. 

The sample and eluents must be filtered through at least a 0.45 pm filter, and the eluents degassed. 

Many different buffers are suitable for use in RP-HPLC, and some of the properties of commonly used buffers are summarized in Table 3. They are added to the eluent prior to pH adjustment (if required) and degassing. The removal of such buffers from product-containing fractions is straightforward and is described in Subheading 2.4.3. 

The degassing unit removes gases dissolved in the eluent. The gas bubbles released from the effluent may complicate the detection of sample. Aqueous eluents can be degassed by increased temperature, by application of vacuum or by displacing effect of helium. 

This procedure guarantees a reproducible preparation of your mobile phase and avoids problems associated with volume contraction. Always prepare a sufficient amount of mobile phase (approx. 1 1) and degas it. Degassing is possible with helium or the built-in degasser (see Tip No. 5). If you use buffers, you should filter them through a membrane. The eluent container should be well covered in order to avoid dirt contamination. 

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