HPLC for Pharmaceutical Scientists 2007 (Part 17)

Developing fast high-performance liquid chromatography (HPLC) methods can improve work efficiency during research, development, or production of a drug substance or a drug product. HPLC is a key technique in all of these areas. Until recently, analysis times of greater than 30 minutes were common. Modern pharmaceutical R&D, with its high-throughput screening, demands high-throughput methods to deal with the large number of samples. To reduce production cycle time, fast HPLC methods are essential for on-line or at-line process control and for rapid release testing. Consider a GMP laboratory responsible for releasing a single batch of drug substance. ...
Nội dung trích xuất từ tài liệu:
HPLC for Pharmaceutical Scientists 2007 (Part 17)17DEVELOPMENT OF FAST HPLCMETHODSAnton D. Jerkovich and Richard V. Vivilecchia17.1 INTRODUCTIONDeveloping fast high-performance liquid chromatography (HPLC) methodscan improve work efficiency during research, development, or production ofa drug substance or a drug product. HPLC is a key technique in all of theseareas. Until recently, analysis times of greater than 30 minutes were common.Modern pharmaceutical R&D, with its high-throughput screening, demandshigh-throughput methods to deal with the large number of samples. To reduceproduction cycle time, fast HPLC methods are essential for on-line or at-lineprocess control and for rapid release testing. Consider a GMP laboratoryresponsible for releasing a single batch of drug substance. Assuming a run timeof 30 minutes and a total of 12 injections, a run time of 6 hours would berequired to cover system suitability, calibration, and sample analysis. If the runtime were 5 minutes, only 1 hour would be required for the analysis. With theadvent of commercial chromatographic porous media of less than 5 µm andmore recently in the 1- to 2-µm range, analyses times of less than 1–2 minuteshave been demonstrated. Hundreds of samples which required days can nowbe analyzed in less than a day. This chapter will focus on how to optimize iso-cratic and gradient methods for speed without sacrificing resolution. In addi-tion, the implication on selection of column dimensions and media particlesize on the speed of methods development will also be discussed. Reducing chromatographic media particle size allows the number of theo-retical plates per second to be increased. However, due to the resolutionHPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBruttoCopyright © 2007 by John Wiley & Sons, Inc. 765766 DEVELOPMENT OF FAST HPLC METHODSdependence on N1/2, doubling of N will only increase resolution by 21/2. As dis-cussed below, a reduction in particle size can lead to a pressure limitation dueto the inverse dependence of pressure drop to the square of the particle diam-eter and the maximum operating pressure of the chromatograph. The key tooptimizing speed is to maximize selectivity, α. Maximizing selectivity for thecritical separation pairs will allow the shortest column lengths and highestmobile-phase linear velocity. Short columns, 3–10 cm packed with particles inthe 1- to 3-µm range, provide high-speed analyses while maintaining reason-able pressure drop. Due to the fast analysis time of these short columns,method development time can also be shortened. Multiple columns can berapidly screened for optimizing selectivity. Short columns are especially usefulwhen the components to be separated are known. However, when dealing withcomplex samples with unknown components such as forced decomposition orbiological samples, using longer columns may be more judicious to achieveoptimum separation of critical components. After selectivity optimization,the method can be optimized for speed by reducing column length. The dis-cussion in this chapter will focus on optimizing speed of analysis and not onselectivity. The reader is referred to Chapters 4 and 8 on how to optimizeselectivity.17.2 BASIC THEORYTo understand how to optimize a separation for speed, it is worth revisitingsome of the theoretical concepts developed earlier in this text. The analysistime, ta, is the time it takes for all sample components to elute off a column ata certain flow rate and is given by L ta = (1 + k ) (17-1) uwhere L is the column length, u is the linear flow velocity of the mobile phase,and k is the retention factor of the latest-eluting peak. Notice here someobvious ways to increase the speed of analysis: The length of the column canbe shortened, mobile phase can be pumped at a faster flow velocity, and onecan ensure that the retention of sample components is not prohibitively long.Once any of these approaches are attempted, however, it is quickly seen thatother important parameters of the separation are affected, principally the res-olution and the column backpressure. These parameters must be consideredwhen enhancing the speed of analysis. Ideally, the analyst would like to max-imize both resolution and speed of analysis, while remaining within the pres-sure capabilities of the instrument.What is discovered, though, is the inevitableexistence of a trade-off between resolution, analysis time, and backpressure.Resolution can be enhanced if more time is allowed; conversely, analysistime can be shortened, but at the expense of resolution. In addition, bothBASIC THEORY 767resolution and speed are limited by the constraints of the instrumentation.The interrelationship between these factors will be considered, startingwith the most important parameter describing the quality of our separation—resolution.17.2.1 Resolution and Analysis TimeThe practical goal of most separations is not to achieve the greatest resolutionpossible, but rather to obtain sufficient resolution to separate all componentsin the shortest amount of time. To optimize for speed, the starting conditionis that there is a minimum resolution requirement for the separation. Resolu-tion is a function of three parameters: column efficiency, or theoretical plates(N), selectivity (a), and the retention factor (k):  N   a − 1   k2  Rs =   (17-2)  4   a   1 + k2  Selectivi ...