Complex gradients

Gradient shape, i.e., the eluent composition as a function of time, ts usually selected ad hoc to satisfy the requirements of the particular separation. In most cases linear gradients are satisfactory, although certain difficult separations may require exponential or more complex gradients. 

Fig. 9 ELSD chromatogram of the molecular species of bovine milk PC. Underivatized molecular species were separated on a 250 X 4.6-mm column packed with Superspher RP-100 at 75°C. The mobile phase contained 1-propanol, water, iso-octane, and ammonium acetate in a complex gradient. (Reprinted from Ref. 102 with the permission of Chromatographia.)...

There are four basic system types. Type I are basic isocratic systems used for simple, routine analysis in a QA/QC environment often for fingerprinting mixtures or final product for impurity/yield checking. Type II systems are flexible research gradient systems used for methods development, complex gradients, and dial-mix isocratics for routine analysis and standards preparation. They fit the most common need for an HPLC system. Type III systems are fully automated, dedicated systems used for cost-per-test, round-the-clock analysis of a variety of gradient and isocratic samples typical of clinical and environmental analysis laboratories. Type TV systems are fully automated gra-... 

Water is the major offender for column contamination problems. I have diagnosed many problems, which customers have initially blamed on detector, pumps, and injectors, that turned out to be due to water impurities. Complex gradient separations are especially susceptible to water contamination effects. 

Another feature of modern HPLC systems that makes them desirable for both analytical and preparative applications is the complex mobile-phase gradients that they are capable of producing. Many systems come equipped with a pump integrator or controller (computer) that allow a number of different mobile-phase solvents to be simultaneously mixed and delivered to the stationary phase. Since this process is automated, complex gradients used for a particular application are quite reproducible. 

Recent studies on separation optimization showed that accurate control of mobile phase pH was essential to successfully resolve a number of important non-hydrolysate amino acids. With good control of a complex gradient profile the system could resolve a mixture of amino acids including, Asn, Gin, cysteine derivatives carboxymethyl cysteine and pyridylethyl cysteine, and the hydroxylated amino acids hydroxyproline (Hyp) and hydroxylysine (Hyl) as well as the hydrolysate amino acids (4). However, the required precision in the control of eluent pH unnecessarily complicated transfer of the method between laboratories. The method also lacked the ability to separate Orn from the hydrolysate amino acids. The current study demonstrates the utility of quaternary HPLC gradient systems for facilitating methods development and simplifying routine eluent preparation with excellent pH control. 

Sophisticated gradient makers capable of generating complex gradients are now on the market (e.g. LKB). The simplest ones to operate generate the gradient using two variable speed pumps and two buffer reservoirs. The pump speeds are automatically controlled by the device so that the gradient follows any pre-determined curve. One other type, which has been used in this field and can be fairly simply constructed is shown in Fig. 4.4. A number, maybe 9, of... 

As previously mentioned, the RP-HPLC of phenolic compounds is carried out with polar eluents. Simple HPLC systems use one pump that pumps a single solvent or, more frequently, a solvent mixture through the column. This is known as isocratic HPLC. More complex gradient systems use more than one pump governed by a computer and pump changing mobile phases that can involve several solvents. Water is usually one of the components in the mixmre the other components most frequently include methanol, acetonitrile, and, more rarely, tetrahydrofuran, etc. The pH of the eluent is always acidic, and this acidity can be attained by the addition of acids such as acetic, formic, phosphoric, trifluoroacetic, and others. The selection of the acid used is dependent on the specific separation problems that need to be solved. 

Li N, Baskaran H, Dertinger SK, Whitesides GM, Van de Water L, Toner M. Neutrophil chemotaxis in linear and complex gradients of interleukin-8 formed in a microfabricated device. Nat Biotechnol 2002 20 826-30. 

Figure 6b. Analytical separations of a crude glycopeptide complex (gradient). Sample crude antibiotic extract. Column Beckman Ultrasphere ODS, 5 pirn, 4.6 x 150 inm. Mobile phase 27 to 37% acetonitrile in 0.1 M KH2PO4PH 3.2. Flow 1.5 ml/min. Detection 220 nm.

Complex gradients can be obtained without a mixing chamber by mixing independently selected streams of solvents A and B. For this purpose, a solenoid valve selects periodically solvent A or solvent B for the pump feed, as shown in Fig. 5. For the Linton gradient former, two valves are used, one being opened while the other is closed. The eluent flow consists of short variable lengths of... 

One of the most commonly used devices for producing a complex gradient is the... 

The number of different curves is B (Siemens), 9 (Spectra-Physics, LDC), 11 (DuPont, Waters) or infinite (Micromeritics and Perkin-Elraer) as the curvature can be selected continuously. In the last instance, the curvature selected must be read precisely in order to obtain good reproducibility. Although the curves are pre-selected, it is often possible to obtain complex gradient shapes, as at any time the programme can be stopped and a new curve selected. With the Orlita gradient system, only a linear gradient is available. However, an external control is possible. 

Calculation of Retention for Complex Gradient Elution High-Performance Liquid Chromatographic Experiments A Universal Approach... 

A numerical method for the calculation of retention under complex gradient HPLC elution conditions Is presented. The approach Is applicable to virtually any gradient or solvent-solute relationship. 

With the program and derived mathematics, the scientist can calculate the retention times and peak widths for any set of solutes, run under any conditions ranging from Isocratlc to complex gradients with multiple columns. Given this mathematical tool, the problem of determining which chromatographic conditions will achieve the desired separation, within any constraints, becomes the next problem to be approached. The solution to such an "optimization" problem Is not easy, however, the necessary universal mathematical tools are now available for researchers In this area to develop approaches to optimization strategies. 

Effective use of computerization in chromatography is further advancing this science. Detailed information is provided regarding calculation and prediction of retention data for isocratic separations that allow optimization of methodologies. Calculation of retention data for complex gradient elution is also shown. 

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