Applications HPLC examples

Similarly, organic liquids have a variety of applications. For example, hexane, which frequently contains impurities such as aromatic compounds, is used in a variety of applications for extracting non-polar chemicals from samples. The presence of impurities in the hexane may or may not be important for such applications. If, however, the hexane is to be used as a solvent for ultraviolet spectroscopy or for HPLC analysis with UV absorbance or fluorescence detection, the presence of aromatic impurities will render the hexane less transparent in the UV region. It is important to select the appropriate grade for the task you have. As an example, three different specifications for n-hexane ( Distol F , Certified HPLC and Certified AR ), available from Fisher Scientific UK, are shown in Figure 5.5 [10]. You will see that the suppliers provide extra, valuable information in their catalogue. 

Since its creation around 1973, modern high-pressure liquid chromatography (HPLC) has played a dominant role in the analysis of pharmaceuticals. It is used in many different applications for example, in content uniformity assays and stability-indicating methods, for the purity profiles of drug substances, or in the analysis of drug metabolism in animals and humans. The heart of all of these assays is the HPLC column. In this chapter, we will describe the fundamental properties of HPLC columns as well as how these properties influence column performance and separation characteristics in pharmaceutical assays. 

In general, a planar method tends to have fewer sample preparative techniques than either gas chromatography (GC) or HPLC methods. The primary criteria for TLC is that the matrix should not distort or streak the analyte band or spot. One other concern should be the stability of the drug after sample application. For example, vitamin D, is stable on prewetted silica gel but decomposes quickly once the sorbent is dried. 

Fluorescence detectors can give improved selectivity over ultraviolet absorption detectors because fewer compounds fluoresce than absorb (Chapter 16). Sensitivities at least as good as and perhaps better than the UV detector are achieved, depending on the geometry of the excitation source-detector arrangement, the intensity of the source, and the quantum efficiency of the fluorophore. The amper-ometric detector (see Chapter 15) is useful for detecting electroactive substances and has found considerable use in biological applications, for example, in the HPLC separation and detection of trace quantities of catecholamines from the brain. 

Self-assembled structures are supramolecular assemblies of covalent backbones structured through intra- and interchain noncovalent interactions. These secondary structures arise from steric constraints and a network of weak interactions (i.e., hydrogen or Van der Waals bonding, dipole-dipole or amphiphilic interactions). Helical morphologies are stiU rarely represented in these artificial species but the control of the heHx sense, and a better knowledge of the chiral amplification mechanism, is highly desirable due to their potential use in many applications. For example, helically chiral polymers can be used as chiral stationary phases for HPLC or for catalysis. 

For more complex samples with a number of electroactive species to be determined, separation by HPLC, or other methods such as ion chromatography or capillary electrophoresis, followed by coulometric detection is better suited. Its applications in HPLC are usually to oxidizable organic species that cannot be determined by ultraviolet absorption, the standard detection technique in HPLC. Examples for such species include amines and phenols, catecholamines (such as the neurotransmitters adrenaline and... 

These problems have been fully addressed in the context of high performance liquid chromatography, so almost any HPLC sales representative will be able to tell you what components are required for any particular application. For example, PEEK (polyether ether ketone) has excellent chemical resistance, is not particularly expensive, is flexible and is biocompatible. It is, therefore, suitable for flow tubing and other components that will come into contact vfith reagents. However, PEEK is attacked by some chemicals including concentrated acids, bromine, chlorine and fluorine. Titanium is a possibility for these sub-... 

As the term implies, in APCI, analyte molecules are ionized by ion-molecule reactions that take place at atmospheric pressure. A scheme for an APCI source is shown in Fig. 12. This ionization technique shows similarities with ESI in that the samples are sprayed into the source thus, this technique is also very commonly used with HPLC (in fact, APCI allows higher flow rates than ESI). On the contrary, there are significant differences between ESI and APCI. First, in APCI, the samples are sprayed into a heated ionization source (t > 400°C) so that the analyte molecules are vaporized. (This implies that APCI is not suitable for the analysis of thermally labile compounds.) An essential part of the APCI source is a corona discharge in which Oj and Nj molecules are ionized and fiorther react with solvent molecules in the gas phase at atmospheric pressure to form ions that will protonate (or deprotonate) the analyte molecules. (Reminder In classical Cl, ion-molecule reactions also take place in the gas phase, but at lower pressure (1-0.1 Torr).) APCI is widely used for ionization of smaller molecules, such as drugs and their metabolites, pesticides, steroid derivatives, lipids, etc. ESI and APCI are often compared to each other in several applications. For example, for the determination of cyclosporin A in rat plasma, see, e.g., the work by Wang et al. [31]. 

From these early studies, dithiocarbamates and their transition metal complexes soon found a host of applications. For example, as a result of their insoluble nature they are widely used in inorganic analysis (3,4). They can also be used to separate different metal ions by high-performance liquid chromatography (HPLC) (5-10) and capillary gas chromatography (GC) (11, 12), and find use as rubber vulcanization accelerators (13), fungicides (14), and pesticides (15). Concomitant with the development of these applications came a burgeoning interest in their general transition metal chemistry and the characteristics and properties of the complexes formed. 

Examples of the application of HPLC to the analysis of (a) acetaminophen, salicylic acid, and caffeine (b) chlorinated pesticides (c) tricyclic antidepressants and (d) peptides. (Chromatograms courtesy of Alltech Associates, Inc. Deerfield, IL). 

A number of analytical techniques such as FTIR spectroscopy,65-66 13C NMR,67,68 solid-state 13 C NMR,69 GPC or size exclusion chromatography (SEC),67-72 HPLC,73 mass spectrometric analysis,74 differential scanning calorimetry (DSC),67 75 76 and dynamic mechanical analysis (DMA)77 78 have been utilized to characterize resole syntheses and crosslinking reactions. Packed-column supercritical fluid chromatography with a negative-ion atmospheric pressure chemical ionization mass spectrometric detector has also been used to separate and characterize resoles resins.79 This section provides some examples of how these techniques are used in practical applications. 

The HPLC method (7) for the PSP toxins has a variety of applications in both research and in public health monitoring programs. A number of advances in our understanding of the biochemistry of PSP are a direct result of this technique. Following is a brief overview of the HPLC method with a couple of examples of its utility in PSP research. 

An excellent example of PLC applications in the indirect coupling version is provided by the works of Miwa et al. [12]. These researchers separated eight phospholipid standards and platelet phospholipids from the other lipids on a silica gel plate. The mobile phase was composed of methylacetate-propanol-chloro-form-methanol-0.2% (w/v) potassium chloride (25 30 20 10 10, v/v). After detection with iodine vapor (Figure 9.2), each phospholipid class was scraped off and extracted with 5 ml of methanol. The solvent was removed under a stream of nitrogen, and the fatty acids of each phospholipid class were analyzed (as their hydrazides) by HPLC. The aim of this study was to establish a standardized... 

In order to reduce the time-consuming open-column chromatographic processes, conventional methods of hydrocarbon-group-type separation have been replaced by MPLC and HPLC. Flash column chromatography is a technique less commonly applied than open-column version, but several applications have been described [2,24—27]. The common technique version is to use a silica-gel-filled column for example, 230 to 400 mesh 20 X 1 cm column size with a back pressure of 1.5 X 10 Pa of an ambient gas such as nitrogen. Solvents are similar to the ones apphed in the case of open-column chromatography fractionations. 

Several applications of varimax rotation in analytical chemistry have been reported. As an example the varimax rotation is applied on the HPLC data table of... 

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