Retention characteristics

The polymer from which a UF membrane is made does not generally affect the retention characteristics of the membrane. However, the nature of the polymer can affect adsorption of species onto the membrane surface. For example, when processing small volumes of dilute solutions, strong adsorption of the solute can diminish its concentration in both the retentate and in the filtrate, and this can affect the apparent retention calculated from Equation 2. 

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The first use for butyl mbber was ia inner tubes, the air-retention characteristics of which contributed significantly to the safety and convenience of tires. Good weathefing, ozone resistance, and oxidative stabiUty have led to appHcations ia mechanical goods and elastomeric sheeting. Automobile tires were manufactured for a brief period from butyl mbber, but poor abrasion resistance restricted this development at the time. 

Two variations of the technique exists isocratic elution, when the mobile phase composition is kept constant, and gradient elution, when the mobile phase composition is varied during the separation. Isocratic elution is often the method of choice for analysis and in process apphcations when the retention characteristics of the solutes to be separated are similar and not dramaticallv sensitive to vei y small changes in operating conditions. Isocratic elution is also generally practical for systems where the equilibrium isotherm is linear or nearly hnear. In all cases, isocratic elution results in a dilution of the separated produces. 

Membrane Characterization The two important characteristics of a UF membrane are its permeability and its retention characteristics. Ultrafiltration membranes contain pores too small to be tested by bubble point. Direc t microscopic observation of the surface is difficult and unreliable. The pores, especially the smaller ones, usually close when samples are dried for the electron microscope. Critical-point drying of a membrane (replacing the water with a flmd which can be removed at its critical point) is utihzed even though this procedure has complications of its own it has been used to produce a Few good pictures. 

The theoretical treatment given above assumes that the presence of a relatively low concentration of solute in the mobile phase does not influence the retentive characteristics of the stationary phase. That is, the presence of a small concentration of solute does not influence either the nature or the magnitude of the solute/phase interactions that determine the extent of retention. The concentration of solute in the eluted peak does not fall to zero until the sample volume is in excess of 100 plate volumes and, at this sample volume, the peak width has become about five times the standard deviation of the normally loaded peak. 

Multidimensional chromatography has important applications in environmental analysis. Environmental samples may be very complex, and the fact that the range of polarity of the components is very wide, and that there are a good many isomers or congeners with similar or identical retention characteristics, does not allow their separation by using just one chromatographic method. 

Returning now to the subject of the chapter, in addition to appropriate retentive characteristics, a potential stationary phase must have other key physical characteristics before it can be considered suitable for use in LC. It is extremely important that the stationary phase is completely insoluble (or virtually so) in all solvents that are likely to be used as a mobile phase. Furthermore, it must be insensitive to changes in pH and be capable of assuming the range of interactive characteristics that are necessary for the retention of all types of solutes. In addition, the material must be available as solid particles a few microns in diameter, so that it can be packed into a column and at the same time be mechanically strong enough to sustain bed pressures of 6,000 p.s.i. or more. It is clear that the need for versatile interactive characteristics, virtually universal solvent insolubility together with other critical physical characteristics severely restricts the choice of materials suitable for LC stationary phases. 

As a result of its highly polar character, silica gel is particularly useful in the separation of polarizable materials such as the aromatic hydrocarbons and polynuclear aromatics. It is also useful in the separation of weakly polar solute mixtures such as ethers, esters and in some cases, ketones. The mobile phases that are commonly employed with silica gel are the n-paraffins and mixtures of the n-paraffins with methylene dichloride or chloroform. It should be borne in mind that chloroform is opaque to UV light at 254 nm and thus, if a fixed wavelength UV detector is being used, methylene dichloride might be a better choice. Furthermore, chloroform is considered toxic and requires special methods of waste disposal. Silica gel is strongly deactivated with water and thus, to ensure stable retentive characteristics, the solvent used for the mobile phase should either be completely dry or have a controlled amount of water present. The level of water in the solvent that will have significant effect on solute retention is extremely small. The solubility of water in n-heptane is... 

In many analyses, fhe compound(s) of inferesf are found as par of a complex mixfure and fhe role of fhe chromatographic technique is to provide separation of fhe components of that mixture to allow their identification or quantitative determination. From a qualitative perspective, the main limitation of chromatography in isolation is its inability to provide an unequivocal identification of the components of a mixture even if they can be completely separated from each other. Identification is based on the comparison of the retention characteristics, simplistically the retention time, of an unknown with those of reference materials determined under identical experimental conditions. There are, however, so many compounds in existence that even if the retention characteristics of an unknown and a reference material are, within the limits of experimental error, identical, the analyst cannot say with absolute certainty that the two compounds are the same. Despite a range of chromatographic conditions being available to the analyst, it is not always possible to effect complete separation of all of the components of a mixture and this may prevent the precise and accurate quantitative determination of the analyte(s) of interest. 

The power of mass spectrometry lies in the fact that the mass spectra of many compounds are sufficiently specific to allow their identification with a high degree of confidence, if not with complete certainty. If the analyte of interest is encountered as part of a mixture, however, the mass spectrum obtained will contain ions from all of the compounds present and, particularly if the analyte of interest is a minor component of that mixture, identification with any degree of certainty is made much more difficult, if not impossible. The combination of the separation capability of chromatography to allow pure compounds to be introduced into the mass spectrometer with the identification capability of the mass spectrometer is clearly therefore advantageous, particularly as many compounds with similar or identical retention characteristics have quite different mass spectra and can therefore be differentiated. This extra specificity allows quantitation to be carried out which, with chromatography alone, would not be possible. 

Qualitative (identification) applications depend upon the comparison of the retention characteristics of the unknown with those of reference materials. In the case of gas chromatography, this characteristic is known as the retention index and, although collections of data on popular stationary phases exist, it is unlikely that any compound has a unique retention index and unequivocal identification can be effected. In liquid chromatography, the situation is more complex because there is a much larger number of combinations of stationary and mobile phases in use, and large collections of retention characteristics on any single system do not exist. In addition, HPLC is a less efficient separation... 

Unlike gas chromatography, in which the mobile phase, i.e. the carrier gas, plays no part in the separation mechanism, in HPLC it is the relative interaction of an analyte with both the mobile and stationary phases that determines its retention characteristics. Hence, it is the varying degrees of interaction of different analytes with the mobile and stationary phases which determines whether or not they will be separated by a particular HPLC system. 

An advantage of the mass spectrometer as a detector is that it may allow differentiation of compounds with similar retention characteristics or may allow the identification and/or quantitative determination of components that are only partially resolved chromatographicaUy, or even those that are totally unresolved. This may reduce the time required for method development and is discussed in more detail in Chapter 3. 

As in other forms of chromatography, the identification of analytes is effected by the comparison of the retention characteristic of an unknown with those of reference materials determined under identical experimental conditions. 

Note - Unequivocal identification of a total unknown using a single HPLC retention characteristic (or indeed a single retention characteristic from any form of chromatography) should not be attempted. 

If the identity of the analyte is genuinely unknown, a farther problem is encountered. In contrast to GC, there are no HPLC systems, combinations of mobile and stationary phases, that are rontinely used for general analyses. Therefore, there are no large collections of k values that can be nsed. For this reason, retention characteristics are often nsed for identification after the nnmber of possible compounds to be considered has been greatly reduced in some way, e.g. the class of compound involved has been determined by colonr tests or UV spectroscopy. 

A more definitive identification may be obtained by combining retention characteristics with more specific information from an appropriate detector. Arguably, the most information-rich HPLC detectors for the general identification problem are the diode-array UV detector, which allows a complete UV spectrum of an analyte to be obtained as it elutes from a column, and the mass spectrometer. The UV spectrum often allows the class of componnd to be determined but the... 

Simplistically, chromatography can be regarded as the separation of the components of a mixture to allow the identification and/or quantitation of some or all of them. Identification is initially carried out on the basis of the chromatographic retention characteristic. This is not sufficient to allow unequivocal identification because of the possibility of more than one analyte having virtually identical retentions. Further information is usually required from an auxiliary technique - often some form of spectroscopy. 

The most widely used LC detector, and the one which, other than the mass spectrometer, gives the most insight into the identity of an analyte, is probably the UV detector, although a UV spectrum very rarely allows an unequivocal identification to be made. It may allow the class of compound to be identified and this, together with the retention characteristics of the analyte, can provide the analyst with a better indication of the identity of the analyte. In the vast majority of cases, however, identification with complete certainty cannot be achieved. 

How then can this problem be addressed From a chromatographic standpoint, the usual method is to change either the stationary phase or, more usually in the case of HPLC, the mobile phase, and look for a change in the retention characteristics. The change observed is usually more characteristic of a single analyte than is the actual retention on any individual chromatographic single system. Even this, however, does not always provide an unequivocal identification. 

In addition, synthesis of reference compounds and a comparison of HPLC retention characteristics and mass spectral data is often reqnired. 

Resolution A term which indicates the ability of a device/technique to sepa-rate/distingnish between closely related signals. In chromatography, it relates to the ability to separate componnds with similar retention characteristics, and in mass spectrometry to the ability to separate ions of similar m/z ratios. 

Li, J. and Carr, P. W., Retention characteristics of polybutadiene-coated zirconia and comparison to conventional bonded phases, Anal. Chem., 68(17), 2857,... 

Mainwaring, N. J., and Reed, A. R., Permeability and Air Retention Characteristics of Bulk Solid Materials in relation to Modes of Dense-Phase Pneumatic Conveying, Bulk Solids Handling, 7(3) 415-425 (1987)... 

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