Scouting gradients

Run the scouting gradient, then narrow the gradient range so that the earliest peak elutes after t() + dwell time (tD) and the last peak elutes near the final solvent concentration. The gradient steepness should be maintained in the narrowed gradient. 

Using initial scouting gradient experiments for efficient development of final gradient or isocratic methods... 

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

My scouting gradient technique was developed when I had to make separations in a customer s laboratory to sell an HPLC system. I only had a few hours to make a separation to convince the customer that he should consider buying a system. But, it provides useful insight for developing a method to use in your laboratory. 

For illustration purposes, we will take the most common case. We will start with a 15-cm long C18 column, 254 nm, and acetonitrile/water in a scouting gradient. Scouting gradients are run much more rapidly than analytical gradients. A mixture of the compounds to be separated is dissolved in 25% acetonitrile in water. A sample is injected into an HPLC equilibrated in the same mobile phase and a 20-min gradient is run to 100% acetonitrile. 

All of these tend to be rather hit or miss. For unknown compounds or unusual mixtures, they generally are not very successful. It is usually better to approach a separation from a more systematic methods development based on scouting gradients (see Chapter 3). 

Figure 12.3 Systematic methods development standards, (a) Scouting gradient (b) isocratic (c) alpha change (d) pH adjustment (e) standards plus internal standards.

With the advent of column switchers and more reproducible alternative column materials, it is now quite feasible to screen multiple pH values—for example, at high, medium, and low pH—using scouting gradients in order to choose the column and pH at which to perform further optimization experiments. This is a particularly tempting scenario when few or no chemical structures are available for the synthetic by-products or degradation products in the sample, or when samples are particularly complex. Recently there has been considerable development on systems for selection of optimal pH and type of column concomitantly [28]. 

If an adequate separation is not obtained by the use of either the reversed-phase analytical method or the silica method, then other stationary phases are scanned for their separation capability (CN, C8, C4, phenyl, chiral, etc.). Often a scouting gradient can be employed to screen columns by HPLC.7 For reversed-phase systems (aliphatic and CN columns), a gradient of acetonitrile and water can be set up, beginning with 100% water and ending with 100% acetonitrile over approximately 30 column volumes. Of the columns that show some peak separation, the gradient can usually be... 

Dolan, J. W. Starting Out Right, Part VI—The Scouting Gradient Alternative LC-GC, 18 478-487, 2000. 

The scout gradient process is particularly suited to analyses where a large number of analytes need to be separated. The rapid ramping of the mobile phase up to a very strong mobile phase causes the elution of all compounds in a short period of time. A linear gradient should be used since it is the easiest from which to extract the isocratic conditions. 

The method optimization chapter reviews five commonly used techniques for assisting bench chromatographers in method optimization or development eluo-tropic series and solvent strength, triangulation, scout gradient, window diagrams, and brute force. Detailed examples and explanations are presented so that these concepts can readily be put to use. Theory is not discussed. 

Fig. 6. Initial scouting gradient for 1,4-bis[(2-pyridin-2-ylethyl)thio]butane-2,3-diol and three unknown impurities. Conditions 11 min gradient from 10 to 80% acetonitrile buffer was 50 mw HCOOH/HCOONH4.

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