Width chromatography peak

The duty cycle of most mass analyzers is long compared to the chromatography peak width. This mismatch might pose a problem in obtaining representative... 

The peak width at half height (wq.s) is the distance between each side of a peak, measured at half the peak height. The peak width measured at half height has no significance with respect to chromatography theory. 

Recalling that a separation is achieved by moving the solute bands apart in the column and, at the same time, constraining their dispersion so that they are eluted discretely, it follows that the resolution of a pair of solutes is not successfully accomplished by merely selective retention. In addition, the column must be carefully designed to minimize solute band dispersion. Selective retention will be determined by the interactive nature of the two phases, but band dispersion is determined by the physical properties of the column and the manner in which it is constructed. It is, therefore, necessary to identify those properties that influence peak width and how they are related to other properties of the chromatographic system. This aspect of chromatography theory will be discussed in detail in Part 2 of this book. At this time, the theoretical development will be limited to obtaining a measure of the peak width, so that eventually the width can then be related both theoretically and experimentally to the pertinent column parameters. 

Using equation (10), the efficiency of any solute peak can be calculated for any column from measurements taken directly from the chromatogram (or, if a computer system is used, from the respective retention times stored on disk). The computer will need to have special software available to identify the peak width and calculate the column efficiency and this software will be in addition to that used for quantitative measurements. Most contemporary computer data acquisition and processing systems contain such software in addition to other chromatography programs. The measurement of column efficiency is a common method for monitoring the quality of the column during use. 

This equation is based on experience with liquid chromatography of low molecular weight samples displaying single peaks. Its application for the GPC of polymers, however, contains a disadvantage, as it mixes two inseparable properties the retention difference for the separation and the peak width for the contrary effect of band broadening. Such a procedure is acceptable if both effects are accessible for an experimental examination. For the GPC experiment, we do not possess polymer standards, consisting of molecules that are truly monodisperse. Therefore, we cannot determine the real peak width necessary for a reliable and reproducible peak resolution R,. This equation then is not qualified for a sufficient characterization of a GPC column. 

One of the difficulties with any form of chromatography is that a band of solute is dispersed, becoming less concentrated as it travels through the system. The efficiency of the column is a measure of the amount of spreading that occurs. In the chromatogram in Fig. 2.3b, Vr = the retention volume of a solute and wg = the volume occupied by the solute. This is called the peak width, but remember it means a volume, not a length. 

Moment analysis is one of the simplest types of analysis and is useful for measuring the performance of the chromatography. Moments can be used to measure the same things that are measured in ID chromatographic systems these include the first, second, and third moments, which are more accurate than the related peak maximum, peak width, and peak asymmetry. In 2D, however, these values each have a component in each dimension and this can be easily determined in software-based measurement systems. 

Metal complexation — One of the most insidious and widely occurrent sources of analytical variation in IEC is product complexation with metal ions. Most proteins can form complexes with metals, regardless of whether or not they are metalloproteins.1 Participant metal ions can derive from the cell culture production process, purification process buffers, or even stainless steel chromatography systems. Complexation can alter retention times, create aberrant peaks, and substantially increase peak width. To the extent that metal contamination of your sample is uncontrolled, so too will be the performance of your assay. 

When the amount of the sample is comparable to the adsorption capacity of the zone of the column the migrating molecules occupy, the analyte molecules compete for adsorption on the surface of the stationary phase. The molecules disturb the adsorption of other molecules, and that phenomenon is normally taken into account by nonlinear adsorption isotherms. The nonlinear adsorption isotherm arises from the fact that the equilibrium concentrations of the solute molecules in the stationary and the mobile phases are not directly proportional. The stationary phase has a finite adsorption capacity lateral interactions may arise between molecules in the adsorbed layer, and those lead to nonlinear isotherms. If we work in the concentration range where the isotherms are nonlinear, we arrive to the field of nonlinear chromatography where thermodynamics controls the peak shapes. The retention time, selectivity, plate number, peak width, and peak shape are no longer constant but depend on the sample size and several other factors. 

A number of kinetic models of various degree of complexity have been used in chromatography. In linear chromatography, all these models have an analytical solution in the Laplace domain. The Laplace-domain solution makes rather simple the calculation of the moments of chromatographic peaks thus, the retention time, the peak width, its number of theoretical plates, the peak asymmetry, and other chromatographic parameters of interest can be calculated using algebraic expressions. The direct, analytical inverse Laplace transform of the solution of these models usually can only be calculated after substantial simplifications. Numerically, however, the peak profile can simply be calculated from the analytical solution in the Laplace domain. 

In RPC, as in all modes of chromatography, when a peptide is eluted under isocratic conditions, the retention can be expressed in either time, te, or volume, Ve, units. Expressions of the elution time or elution volume of a peptide, eluting with a peak width w (= 4ot=4ov), from a column packed with RPC particles of mean particle diameter dp, incorporate the physical aspects of the column (diameter dc, length L), the flow rate F (or linear flow velocity, v = LFIVm) and the phase ratio, mobile phase in the chromatographic column. Usually, retention dependencies for a peptide P, are represented in terms of a capacity factor k as follows ... 

The shape ofthe elution curve for a pulse injection can be approximated by the Gaussian error curve for AT > 100, which is almost the case for column chromatography [2]. The value of N can be calculated from the elution volume Vg (m ) and the peak width W (m ), which is obtained by extending tangents from the sides of the elution curve to the baseline and is equal to four times the standard deviation (Ty (m ) = as shown in Figure 11.7. 

A chromatography column of 10 mm i.d. and 100 mm height was packed with particles for gel chromatography. The interparticle void fraction e was 0.20. A small amount of a protein solution was applied to the column and elution performed in an isocratic manner with a mobile phase at a flow rate of 0.5 cm min. The distribution coefficient A of a protein was 0.7. An elution curve of the Gaussian type was obtained, and the peak width W was 1.30 cm . Calculate the Hs value of this column for this protein sample. 

A 1.0 cm-i.d. and 50 cm-long chromatography column is packed with gel beads that are 100 pm in diameter. The interparticle void fraction e is 0.27, and the flow rate of the mobile phase is 20 cm h. A retention volume of 20 cm and a peak width W of 1.8 cm were obtained for a protein sample. 

In chromatography separations operated under gradient elution, one simple method of obtaining an equal peak width of a specific solute in columns of different dimensions is to keep the numbers of theoretical stages of both columns equal. 

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