Instrumentation HPLC separation technologies

By examining this brief history of the development of instrumentation and methods, it should be clear what parameters define the instrument of choice for analysis of pharmaceutically important molecules. Such an instrument is one capable of performing LC-MS, typically using a reverse-phase HPLC separation and one of the API techniques. MS-MS capability is desirable—using an ion trap, a triple quadrupole, or other tandem mass analyzer instrument. Accurate mass measurement capability is also desirable. These attributes make up a prioritized list of capabilities for the ideal full-purpose instrument to be used in a pharmaceutical research environment. As one progresses through this list, the expense of the instrument increases, the sophistication of the instrument increases, and the intellectual and technological commitment required to do these experiments increases. 

Further advances in HPLC instrumentation and column technology were made in 2004, with significant increases to the resolution, speed, and sensitivity in liquid chromatographic separations. Columns packed with smaller particles (1.7 mm) and instrumentation with specialized capabilities designed to deliver mobile phase at 15,000 psi (1000 bars) were needed to achieve a new level of performance. UPLC... 

CE instrumentation is quite simple (see Chapter 3). A core instrument utilizes a high-voltage power supply (capable of voltages in excess of 30,000 V), capillaries (approximately 25—lOOpm I.D.), buffers to complete the circuit (e.g., citrate, phosphate, or acetate), and a detector (e.g., UV-visible). CE provides simplicity of method development, reliability, speed, and versatility. It is a valuable technique because it can separate compounds that have traditionally been difficult to handle by HPLC. Furthermore, it can be automated for quantitative analysis. CE can play an important role in process analytical technology (PAT). For example, an on-line CE system can completely automate the sampling, sample preparation, and analysis of proteins or other species that can be separated by CE. 

Several technology leaps have taken place in separation sciences during the lifetime of the pharmaceutical industry. The development of chromatography at the end of the nineteenth century was the first of these revolutions and its transformation into thin-layer chromatography (TLC) provided the mainstay for quantitative analysis well into the second half of the twentieth century. With the development of gas chromatography (GC) after World War II and high-performance liquid chromatography (HPLC) two decades later, the age of fully instrumented separation science had arrived. 

Capillary electrochromatography (CEC) is a rapidly emerging analytical separation technique, with several different instrumental formats and prepacked CEC capillary columns now available. P15-323 As an advanced nanoseparation technology, CEC represents an orthogonal hybrid of HPLC and HP-CZE. As a consequence, resolution can be achieved... 

HPLC is becoming by far the most popular technique for the separation of flavonoids, both on preparative and analytical scales. Improvements in instrumentation, packing materials, and column technology are being introduced all the time, making the technique more and more attractive. 

Figure 3.1—Schematic of an HPLC system and example of a modular commercial HPLC. A modular system allows users to tailor the instrument to their needs and budget. The HP 1100 chromatograph equipped with an autosampler and a mass detector that allows continuous operation is shown (reproduced by permission of Agilent Technologies). Regulation of the temperature of the column greatly improves the reproducibility of the separation.

The advances in column and instrument technology have significantly enhanced HPLC performance in recent years. Results comparing the effects of various column packings on TG separation by RP-HPLC were presented by El-Hamdy and Perkins (87). Six commercially packed columns produced by different manufacturers were used PARTISIL ODS-1 and ODS-2 octadecyl-bonded silica of 10-/rm partical size, ZORBAK-ODS octadecyl-silica of 6-7-/rm diameter (250 X 4.6-mm ID), 5-/rm octyl-bonded spherical silica LC-8, 5-//m methyl-bonded spherical silica LC-1, and a 5-/rm octadecyl-bonded spherical silica LC-18 (150 X 4.6-mm ID). The mobile phase employed consisted of mixtures of methanol/acetone/isopropanol/acetonitrile ranging from l 0 3 4to 1 6 3 4. Triglycerides were solubilized in either THF or acetone at 100 mg/ml for each compound. 

Separation speed and ease of use seem to be the primary factors driving changes in HPLC instrumentation. Resolution efficiency and stationary phase stability, especially at high pH, are the primary factors affecting current changes in column technology. 

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