Solvents for HPLC Operation

Occasionally, a single solvent does not provide suitable resolution of solutes. Solvent binary mixtures can be prepared with eluent strengths intermediate between the e° values for the individual solvents. 

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Solvent Reservoirs. Storage of sufficient amount of HPLC solvents for continuous operation of the system. Could be equipped with an online degassing system and special filters to isolate the solvent from the influence of the environment. 

The flexibility of TLC relative to HPLC is enhanced by the greater choice of solvents available for preparing TLC mobile phases. The choice of solvents for HPLC is limited by the requirements for their chemical and physical properties imposed by the nature of the method. HPLC is a closed system operated under high pressure with on-line detection, most often using a UV monitor, and the column is continually reused. Solvent components with high vapor pressure (e.g., ethyl ether) or UV absorbance (benzene) or those that might degrade the column (NaOH) are difficult to use in HPLC but are readily applicable to TLC. 

LC-NMR can be operated in two different modes on-flow and stopped-flow. In the onflow mode, LC-NMR spectra are acquired continuously during the separation. The data are processed as a two-dimensional (2D) NMR experiment. The main drawback is the inherent low sensitivity. The detection limit with a 60 p.1 cell in a 500 MHz instrument for a compound with a molecular weight around 400 amu is 20 pig. Thus, on-flow LC-NMR runs are mainly restricted to the direct measurement of the main constituents of a crude extract and this is often under overloaded HPLC conditions. Typically, 1 to 5 mg of crude plant extract will have to be injected on-column.In the stopped-flow mode, the flow of solvent after HPLC separation is stopped for a certain length of time when the required peak reaches the NMR flow cell. This makes it possible to acquire a large number of transients for a given LC peak and improves the detection limit. In this mode, various 2D correlation experiments (COSY, NOESY, HSQC, HMBC) are possible. 

The fraction of column effluent containing the analyte and internal standard can be either collected manually for subsequent reinjection onto the second (analytical) column (offline operation) or diverted directly onto the second column via a high-pressure switching valve (online operation). For manual collection, a drop-counter-fraction collecting system rather than a volume collection system has been recommended (117). The fraction is collected in a small tapered tube, and the solvent is carefully evaporated off under a stream of nitrogen. The residue is then dissolved in a small volume of a suitable solvent for the analytical separation. Because the sample is reconstituted in offline operation, the potential problem of mobile phase incompatibility between the two HPLC systems is avoided, and hence any semipreparative/analytical combination can be used. 

Equipment for recycle operations differs from conventional HPLC equipment. For a recycle system to be useful, the extracolumn band spreading must be small relative to the band spreading of the column. This involves the solvent delivery system, transport tubing, and detector(s). Also, because a recycle system is a closed system with a finite volume, the operator must be aware that fast-moving materials could eventually overtake slower-moving materials and remix. To prevent peak overlap, a means must be provided to allow the operator to remove a portion of the sample components before overlap can occur. 

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