Developing Gradient Separations

For more on developing gradient separations, see reference 1 and J. W. Dolan, The Scouting Gradient Alternative. LCGC 2000,18, 478. 

The column used for blood serum analysis was 100 cm long, 1 mm in diameter and packed with RP 18 reversed phase having a particle size of 10 pm. A concave gradient program was used to develop the separation over a period of 45 min. at a flow rate of 50 pl/min. The initial solvent was 75% methanol 25% water and the final solvent was pure methanol. 

Finally, it is worth emphasizing once again that the C8 reverse phase, with a 3 p particle size, packed in a column 3 cm long and 4.6 mm in diameter is an excellent scouting column. A column of this size can be made to provide very rapid separations and subsequently can be quickly reconditioned to another mobile phase. By using such a column, and employing a gradient from pure water to pure acetonitrile to develop the separation, the complexity of the sample will often be revealed, and from the results an improved phase system can be educed. 

Chromatography. A number of HPLC and TLC methods have been developed for separation and isolation of the brevetoxins. HPLC methods use both C18 reversed-phase and normal-phase silica gel columns (8, 14, 15). Gradient or iso-cratic elutions are employed and detection usually relies upon ultraviolet (UV) absorption in the 208-215-nm range. Both brevetoxin backbone structures possess a UV absorption maximum at 208 nm, corresponding to the enal moeity (16,17). In addition, the PbTx-1 backbone has an absorption shoulder at 215 nm corresponding to the 7-lactone structure. While UV detection is generally sufficient for isolation and purification, it is not sensitive (>1 ppm) enough to detect trace levels of toxins or metabolites. Excellent separations are achieved by silica gel TLC (14, 15, 18-20). Sensitivity (>1 ppm) remains a problem, but flexibility and ease of use continue to make TLC a popular technique. 

Selectivity of the separation in TLC is achieved by various of the aforementioned techniques (e.g. multiple development, gradient elution, sequence TLC, AMD, HPPLC or OPLC). Multidimensional TLC methods are described in Section 7.4.4. 

Method development is vastly simplified by computer simulations using commercial software. With input from a small number of real experiments, a program can predict the effects of solvent composition and temperature in isocratic or gradient separations. You can select optimum conditions in minutes with the computer instead of days in the lab. Of course, you must verify the prediction by a real experiment. Commercial software saves huge expenses in method development in industrial laboratories. 

A methanol-water rate gradient was developed which separated the standard BaP metabolites (received from the National Cancer Institute Chemical Carcinogen Reference Standard Repository, a function of the Division of Cancer Cause and Prevention, NCI, Bethesda, MD 20205) and re-equilibrated the column in about 30 min. A diagram of the gradient is shown in Figure 1. A mixture of BaP metabolite standards was separated using this system and eluted in the following order 7,8-diol-9,10-epoxide and... 

To develop a gradient separation, the appropriate strengths for the different eluents must be determined. Often the best approach is to start with a linear binary gradient, beginning with 100% of the weak eluent (e.g., water) and ending with 100% (e.g., acetonitrile, ACN) of the strong eluent. However, the two solutions must be completely miscible at the concentrations used. 

Hardware contributions to gradient performance High-pressure or low-pressure gradient formation Mixing devices Gradient precision Developing a gradient separation The three basic situations... 

Early components bunched, late components bunched Early components bunched, late components resolved Early components resolved, late components resolved Applying a strategy for developing a gradient separation Effect of flow rate upon gradient performance Applications Practical considerations... 

Developing a gradient separation involves varying the percent change in mobile phase composition per unit volume of mobile phase delivered. This rate of change (ROC) in solvent composition per unit volume is graphically represented in Figure 7-7. As the slope of the line is decreased, the resolution of the separation will improve until the separation is limited by the efficiency of the column. The ROC value can be calculated by ... 

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