HPLC instrumentation detectors

Ion-exchange columns can be substituted into the general HPLC instrument shown in Eigure 12.26. The most common detector measures the conductivity of the mobile phase as it elutes from the column. The high concentration of electrolyte in the mobile phase is a problem, however, because the mobile-phase ions dominate the conductivity, for example, if a dilute solution of HCl is used as the mobile phase, the presence of large concentrations of H3O+ and Ck produces a background conductivity that may prevent the detection of analytes eluting from the column. 

Size-exclusion chromatography can be carried out using conventional HPLC instrumentation, replacing the HPLC column with an appropriate size-exclusion column. A UV/Vis detector is the most common means for obtaining the chromatogram. 

Other properties of solvents which need to be considered are boiling point, viscosity (lower viscosity generally gives greater chromatographic efficiency), detector compatibility, flammability, and toxicity. Many of the common solvents used in HPLC are flammable and some are toxic and it is therefore advisable for HPLC instrumentation to be used in a well-ventilated laboratory, if possible under an extraction duct or hood. 

FIA has also found wide application in pharmaceutical analysis.214,215 Direct UV detection of active ingredients is the most popular pharmaceutical analysis application of FIA. For single component analysis of samples with little matrix interference such as dissolution and content uniformity of conventional dosage forms, many pharmaceutical chemists simply replace a column with suitable tubing between the injector and the detector to run FIA on standard HPLC instrumentation. When direct UV detection offers inadequate selectivity, simple online reaction schemes with more specific reagents including chemical, photochemical, and enzymatic reactions of derivatization are applied for flow injection determination of pharmaceuticals.216... 

The mobile phase in HPLC is called the eluent and is a liquid or a mixture of liquids. Common eluents are water, aqueous solutions, acetonitrile, and methanol. Almost any other common solvent compatible with the column packing and the detector may be used. In some cases, the HPLC instrument will be capable of making a mixture of eluents or changing the mixture of eluents during chromatography. If this is done, care must be taken to make sure that the eluent mixture is compatible with the detector. 

A simple system is comprised of an isocratic pump, a manual injector, a UV detector, and a strip-chart recorder. A schematic diagram of an HPLC instrument is shown in Fig. 15.4. This simple configuration is rarely used in most modern laboratories. A typical HPLC system is likely to consist of a multi-solvent pump, an autosampler, an on-line degasser, a column oven, and a UV/Vis or photodiode array detector all connected to and controlled by a data-handling workstation. Examples of modular and integrated systems are shown in Fig. 15.5. Some of the important instrumental requirements are summarized in Table 15.2. 

Type of detector Supplier Detection port no HPLC instrument part no... 

The basic theory, principles, sensitivity, and application of fluorescence spectrometry (fluorometry) were discussed in Chapter 8. Like the UV absorption detector described above, the HPLC fluorescence detector is based on the design and application of its parent instrument, in this case the fluorometer. You should review Section 8.5 for more information about the fundamentals of the fluorescence technique. 

Examine the HPLC instrument to which you are assigned. Find the inlet line to the pump and place the free end of this line in the reservoir containing the mobile phase with the 90/10 composition. Trace the path of the mobile phase from the reservoir, through the pump, injection valve, column, and detector, to the waste container so that you identify and recognize all components of the flow path. Turn on the pump and detector and begin pumping the mobile phase at a rate... 

The traditional HPLC instrument is composed of two different parts the first part separates the components of the sample and the other part accomplishes the detection of the components separated. The part of the HPLC carrying out the separation contains a column, an injection device and the eluent delivery system (pump with filters, degasser and transfer tubing, eventually a mixer for gradient elution). One or more detectors, a signal output device coupled with appropriate software, are responsible for detection and primary data evaluation. Pumps deliver the eluent or the different components of the eluent into the column with a precise, constant and reproducible flow rate. 

HPLC instrument equipped with a UV-vis detector column chromatography equipment. 

This chapter presents an overview of current trends in high-pressure liquid chromatography (HPLC) instrumentation focusing on recent advances and features relevant to pharmaceutical analysis. Operating principles of HPLC modules (pump, detectors, autosampler) are discussed with future trends. 

However, those who have worked with smaller diameter columns have often experienced lower performance and other difficulties. This is primarily due to extra-column effects the bandspreading in the injector, detector and tubing, or the gradient delay volume of the instrument. Troubleshooting guidelines for sorting out the causes of these difficulties are available in Reference f. With proper care, 2-mm columns can be run on a standard modern HPLC instrument with few difficulties. Smaller i.d. columns require special instrumentation. 

The promise of monolith is the achievement of a higher performance at a lower backpressure than a packed bed. While this is true in principle, current implementations are limited by the fact that the external wall to the structure is made from PEEK. At the time of this writing, the commercially available monoliths can only be used up to a pressure of 20MPa (200 atm, 3000 psi), while packed bed steel columns can be used up to double this pressure and higher. Also, the preparation of the monolith appears to be cumbersome. At the current time, the silica-based monoliths are available only with an internal diameter of 4.6mm. The speed is thus also limited by the flow rate achievable by the HPLC instrument. At the same time, the detector of choice today is the mass spectrometer, which can tolerate only much... 

HPLC methods can usually be transferred without many modifications, since most commercially available HPLC instruments behave similarly. This is certainly true when the columns applied have a similar selectivity. One adaptation, sometimes needed, concerns the gradient profiles, because of different instrumental or pump dead-volumes. However, larger differences exist between CE instruments, e.g., in hydrodynamic injection procedures, in minimum capillary lengths, in capillary distances to the detector, in cooling mechanisms, and in the injected sample volumes. This makes CE method transfers more difficult. Since robustness tests are performed to avoid transfer problems, these tests seem even more important for CE method validation, than for HPLC method validation. However, in the literature, a robustness test only rarely is included in the validation process of a CE method, and usually only linearity, precision, accuracy, specificity, range, and/or limits of detection and quantification are evaluated. Robustness tests are described in references 20 and 59-92. Given the instrumental transfer problems for CE methods, a robustness test guaranteeing to some extent a successful transfer should include besides the instrument on which the method was developed at least one alternative instrument. 

Apparatus. The HPLC instrument used was a Water s Associates model 6000A pump for the solvent supply, a U6K septumless injector and a radial compression module with standard Radial Pak columns. Immediately after the column a low dead volume tee was inserted and another 6000A pump was used to deliver a solution of OPT for the post-column derivatization of histamine. Twenty feet of 9 thousandths (id) coiled stainless steel tubing was used as a mixing chamber and held at 60 C in a water bath. The reaction mixture then passed through a Water s 420 fluorescence detector which was connected to a recorder. The detector was equipped with a 340-nm excitation filter and a 440-nm emmission filter. 

High-performance liquid chromatography (HPLC) is one of the premier analytical techniques widely used in analytical laboratories. Numerous analytical HPLC analyses have been developed for pharmaceutical, chemical, food, cosmetic, and environmental applications. The popularity of HPLC analysis can be attributed to its powerful combination of separation and quantitation capabilities. HPLC instrumentation has reached a state of maturity. The majority of vendors can provide very sophisticated and highly automated systems to meet users needs. To provide a high level of assurance that the data generated from the HPLC analysis are reliable, the performance of the HPLC system should be monitored at regular intervals. In this chapter some of the key performance attributes for a typical HPLC system (consisting of a quaternary pump, an autoinjector, a UV-Vis detector, and a temperature-controlled column compartment) are discussed [1-8]. 

The method of complete electrolysis is also important in elucidating the mechanism of an electrode reaction. Usually, the substance under study is completely electrolyzed at a controlled potential and the products are identified and determined by appropriate methods, such as gas chromatography (GC), high-performance liquid chromatography (HPLC), and capillary electrophoresis. In the GC method, the products are often identified and determined by the standard addition method. If the standard addition method is not applicable, however, other identification/determination techniques such as GC-MS should be used. The HPLC method is convenient when the product is thermally unstable or difficult to vaporize. HPLC instruments equipped with a high-sensitivity UV detector are the most popular, but a more sophisticated system like LC-MS may also be employed. In some cases, the products are separated from the solvent-supporting electrolyte system by such processes as vaporization, extraction and precipitation. If the products need to be collected separately, a preparative chromatographic method is use-... 

Most HPLC instruments are on line with an integrator and a computer for data handling. For quantitative analysis of HPLC data, operating parameters such as rate of solvent flow must be controlled. In modern instruments, the whole system (including the pump, injector, detector, and data system) is under the control of a computer. 

It is imperative that the HPLC instrument, including the detector, is working correctly. The easiest way to check this is by first running a blank. If there is no response, one can move onto injecting the standards. If there is a response to the blank, the column may have been overloaded prior to this run. Refer to a troubleshooting guide for the specific HPLC system. The internet is also an invaluable source for troubleshooting (e.g., see Internet Resources). Keep in mind that the source of the problem may not be the system but may in fact be the column. 

Similar to GC instruments, HPLC instruments consist of an injection port, a separation column, a detector, and an instrument control/data acquisition computer. The use of liquid as a mobile phase influenced the design and construction materials of HPLC instrumentation elements. A sample extract or an aqueous sample is introduced into the separation column through an injection loop that can be programmed to receive various volumes of liquid (5 pi to 5 ml). 

Commercially available HPLC instrumentation was originally designed for use with standard-bore columns (4.6 mm I.D.). Detector flow cells were optimized for maximum sensitivity with these analytical columns, injectors were designed to introduce microliter quantities of sample, and pumps were designed to be accurate and reproducible in the milliliter flow-rate ranges commonly employed with standard-bore columns. However, these instruments are not well suited for use with small-bore columns, as the dispersion introduced by the large volumes is detrimental to the separation. In addition, the reproducibility and accuracy of the pumping system at the low flow rates required are questionable. 

With a low-dose dmg product, the absorptivity of dmg substance is a key property. If it is intense, conventional detection methodology may be viable, typically with a UV-Visible detector using HPLC instrumentation. However, LC/MS may be another option when increased sensitivity is needed.23... 

For the determination of the concentrations of the light and heavy phases a modified HPLC instrument from Carlo Erba (System 200) was used as an SFC instrument. The columns used were a 10 mm pre column and a 60 mm main column of 2 mm inner diameter packed with Spherisorb (SI 3 p). Detection was carried out with an UV detector at a wavelength of 284 nm. 

Data Acquisition and Control System. Computer-based system that controls all parameters of HPLC instrument (eluent composition (mixing of different solvents) temperature, injection sequence, etc.) and acquires data from the detector and monitors system performance (continuous monitoring of the mobile-phase composition, temperature, backpressure, etc.). 

---