Peaks resolution

The resolution between neighboring peaks is another common optimization goal. There are two approaches to influence resolution in chromatography  

Optimizing the resolution enhances the accuracy and precision of quantitative analysis and can also improve method robustness. It can even reduce the amount of sample preparation provided that the optimized method can handle a direct injection of samples with more complex matrices. Improved separation efficiency also translates into a higher number of components that the method is able to separate, the so-called theoretical peak capacity. Economical benefit may result from simphfied workflows with less sample preparation and improved data quality to account for more accurate analyses. 

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Elution volume, exclusion chromatography Flow rate, column Gas/liquid volume ratio Inner column volume Interstitial (outer) volume Kovats retention indices Matrix volume Net retention volume Obstruction factor Packing uniformity factor Particle diameter Partition coefficient Partition ratio Peak asymmetry factor Peak resolution Plate height Plate number Porosity, column Pressure, column inlet Presure, column outlet Pressure drop... 

An analytical method vahdation study should include demonstration of the accuracy, precision, specificity, limits of detection and quantitation, linearity, range, and interferences. Additionally, peak resolution, peak tailing, and analyte recovery are important, especially in the case of chromatographic methods (37,38). 

For SEC separations of polymers, column efficiency is better characterized by specific resolution, R,p, and efficiency, T, than by theoretical plate count. Peak resolution, R is calculated according to (7) ... 

Unfortunately, this value is sample and system dependent just by using polymer standards with different molar masses, different values of peak resolution can be generated. 

A sample independent parameter is the so-called specific resolution, R,p, which can be obtained from the peak resolution by normalizing the values according to the molar masses of the samples used for testing (24) ... 

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. 

TABLE 21.4 Peak Resolution Parameters Valley-to-Peak Ratios, v, and Peak Separation Parameters, P, for Batch 52 Gel... 

Plate number N (as for Gaussian peaks) Resolution factor R ... 

The mercury film electrode has a higher surface-to-volume ratio than the hanging mercury drop electrode and consequently offers a more efficient preconcentration and higher sensitivity (equations 3-22 through 3-25). hi addition, the total exhaustion of thin mercury films results in sharper peaks and hence unproved peak resolution in multicomponent analysis (Figure 3-14). 

In a further study, Rill et al. [325] developed a model of gel permeation chromatography that included a bimodal pore stracture. The smallest mode in the pore-size distribution represents the basic background polyacrylamide pore structure of about 1-mn mean radius, and the second mode was around 5 nm, i.e., in the range of size of the molecular templates. The introduction of this second pore structure was found to substantially improve the peak resolution for molecules with molecular sizes in the range of the pore size. 

While earlier papers cited buffer systems or aqueous o-phosphoric acid to achieve satisfactory peak resolution, most recent investigations involved acetic acid or formic acid systems. " Representative examples are 0.2% and 1% HCOOH for betacyanins and betaxanthins, respectively, the latter requiring a lower pH for chromatographic resolution. Methanol or acetonitrile are most commonly used as modifiers, either undiluted or diluted with purified water at ratios of 60 40 or 80 20 (v/v), respectively. - Typical HPLC fingerprints for yellow and red beet juice are shown in Figure 6.4.1. 

Optimized HPLC separation allows most betaxanthins to be separated on a Cl8 reversed phase stationary phase according to their respective polarities. - Considerable progress was achieved by the introduction of a highly polar silica-based column, which allowed major improvement of peak resolution, especially at early... 

Peedc-to-peak resolution in SEC can be calculated by the ratio of peak separation at the peak maxiaut to the sum of the baseline peak widths. This general definition of resolution is less useful in SEC, where a measure of the ability of the column to separate solutes of different molecular weight is required. For this purposes, we define a new term, the specific resolution factor, R, which relates peak resolution to sample molecular weight, assuming all measurements are made within the linear region of the molecular weight calibration curve, equation (4.41)... 

The modem HPLC system is a very powerful analytical tool that can provide very accurate and precise analytical results. The sample injection volume tends to be a minor source of variation, although fixed-loop detectors must be flushed with many times their volume in sample to attain high precision. Assuming adequate peak resolution, fluorimetric, electrochemical, and UV detectors make it possible to detect impurities to parts per billion and to quantitate impurities to parts per thousand or, in favorable cases, to parts per million. The major sources of error in quantitation are sample collection and preparation. Detector response and details of the choice of chromatographic method may also be sources of error. 

Rawjee, Y. Y and Vigh, Gy., A peak resolution model for the capillary electrophoretic separation of the enantiomers of weak acids with hydroxypropyl (3-cyclodextrm-containing background electrolytes, Anal. Chem., 66, 619, 1994. 

Purged packed Packed Large-bore capillary N/a N/a Reasonable if good peak resolution is not required... 

The above conclusions clearly indicate that the use of very thin mercury films gives maximal sensitivity as it enables fast sweep rates to be combined with excellent peak resolution. 

Felinger, A. (1997). Critical peak resolution in multicomponent chromatograms. Anal. Chem. 69, 2976. 

As with any separation technique, the desired goal is to maximize peak resolution at the fastest speed. Higher resolution in 2DLC is easier to achieve than when using onedimensional chromatography because selectivity differences between the two different columns can give a resolution enhancement. This is easily seen through the simplified resolution equation, discussed in Chapter 2,... 

In summary such marked structural differences can be assumed to affect the structure of the initial nanoparticles in the solidification process. Improved peak resolution, deconvolution, Q1 ratio-possible ring structure matching, model calculations, and comparison with NMR and other structural data can make Raman a quite useful, inexpensive, and mobile accessory for studying the structure of silicates both in aqueous solutions and in solid phase. 

Buffer parameters such as type of buffer used, its pH and ionic strength, and organic solvent content can be varied to influence the electrophoretic mobility of target compounds leading to an improved peak resolution. 

Often, to increase peak resolution and capacity for a complex sample mixture, the term hyphenation can be referred also to different chromatographic modes coupled together. 

Rawjee, Y.Y., Williams, R.L., Vigh, G. Capillary electrophoretic chiral separations using cyclodextrin additives III. Peak resolution surfaces for ihuprofen and homatropine as a function of the pH and the concentration of fS-cyclodextrin. J. Chromatogr. 1994, 680, 599-611. 

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