HPLC operation

In recent years, much effort has been devoted to developing HPLC-MS methods for natural compounds. The main difficulty lies in the different operating conditions of HPLC and MS. While HPLC operates using high flow rates, high pressures, and liquid phases near room temperature, MS uses low flow rates, high vacuum, and a gas phase ion analyzer. Various interfaces allow for online coupling of these two... 

Table 3 HPLC operating conditions for the determination of anilides... 

HPLC and LC/MS. HPLC methodology coupled with ultraviolet (UV), fluorescence (FL), photodiode-array (PDA) and/or a mass spectrometry (MS) detection has been developed. In general, neonicotinoids can be determined by HPLC/UV. Typical HPLC operating conditions are given in Table 2. 

B. HPLC operating conditions for confirmatory analysis on the Quattro II triple-quadrupole mass spectrometer... 

Mix effluents of HPLCs (performed by user) Mix effluents of several HPLC columns before introducing to MS HPLCs operated in parallel may or may not be simultaneous... 

Multiple sprayer ion source (performed by instrument vendor or advanced user) Purchase or build LC-MS interface containing two or more sprayers in same interface housing each sprayer receives effluent from HPLC column HPLCs operated in parallel and simultaneously... 

Staggered parallel chromatography (performed by user or commercial supplier) Effluents from two to four HPLCs fed to multiposition valve sprayer of MS receives effluent from only one HPLC at a time HPLCs operated in parallel and offset from each other... 

This book s focus is on analytical scale HPLC, so preparative scale LC and micro LC are not discussed in detail here. In-depth treatments of these techniques are found elsewhere.LC/MS, data handling and practical HPLC operation are discussed in later chapters. 

HOW TO BE MORE SUCCESSFUL WITH HPLC ANALYSIS PRACTICAL ASPECTS IN HPLC OPERATION... 

This chapter describes how to be more successful in HPLC operation by summarizing a series of standard operating procedures representing the best practices of experienced HPLC analysts. It offers some brief guidelines on maintenance/troubleshooting and on means of enhancing HPLC assay precision. 

The following is a summary of important procedures or sequence of events for HPLC operation ... 

This section contains a list of steps the analyst can take to ensure successful HPLC operation and includes highlights of key maintenance and troubleshooting strategies. 

Micro-HPLC operation sets special demands on the gradient instrumentation. As the internal column diameter, d, decreases, lower flow rates should be used at comparable mean linear mobile phase velocities, u = 0.2-0.3 mm/s. At a constant operating pressure, the flow rate decreases proportionally to the second power of the column inner diameter, so that narrow-bore LC columns with 1mm i.d. require flow rates in the range of 30-100pL/min, micro-columns with i.d. 0.3-0.5mm, flow rates in between 1 and lOpL/min, and columns with 0.075-0.1 mm i.d. flow rates in the range of hundreds nL/min. Special miniaturized pump systems are required to deliver accurately mobile phase at very low flow rates in isocratic LC. 

Until recently, HPLC operated at pressures of —7-40 MPa (70-400 bar, 1 000-6 000 pounds/inch2) to attain flow rates of —0.5-5 mL/min. In 2004, commercial equipment became available to employ 1.5- to 2-pm-diameter particles at pressures up to 100 MPa (1000 bar, 15 000 pounds/inch2). These instruments enable a substantial improvement in resolution or a decrease in run time. Table 25-1 shows theoretical performance for different particle sizes such performance was realized in research with ultrahigh-pressure equipment. 

Sample Preparation and Selection of HPLC Operating Conditions... 

In certain cases, affinity columns can be used to fractionate within classes of bound materials for instance, protein A antibody columns have been used to separate the various subtypes of IgG. In this case, the packings are micro-porous, heavily cross-linked polymers and benefit from HPLC operating conditions. Eluting conditions are usually step gradients of buffers with different pHs. The last step of a protein G column is at a very acidic pH and the sample is eluted into a buffer solution that quickly raises the pH to prevent protein denaturation. 

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