Chromatographic influence

A chromatographic column provides a location for physically retaining the stationary phase. The column s construction also influences the amount of sample that can be handled, the efficiency of the separation, the number of analytes that can be easily separated, and the amount of time required for the separation. Both packed and capillary columns are used in gas chromatography. 

The influence of NH., and CO, on the chromatographic behaviour of benzoic acid and its derivatives (o-, m-, p-hydroxybenzoic, nitrobenzoic, aminobenzoic, chlorobenzoic acids) was studied. The work was carried out by means of upgoing TLC on Sorbfil plates. Isopropanol- and ethyl acetate-containing water-organic eluents were used as mobile phases in the absence or presence of gaseous modifiers in the MP. The novel modification of TLC has been found to separate benzoic acids with different values of their dissociation constants more effectively than water-organic mobile phases. 

Methods which ai e described in Phamiacopoeias (American, British, and European) ai e based on using narrow standai ds for calibration and broad standai d for system suitability test. Prescribed limits of system suitability ar e broad and therefore it may cause large uncertainty of results. But on the other side results ar e strongly influenced by par ameters of chromatographic system. 

To eluate an antibiotic from chromatographic columns as a mobile phase such solvents as methanol, ethanol, propanol, acetone, acetonitrile are usually used. Influence of these solvents on ions Eu(III) in a complex with OxTC and Cit has been investigated. It is established, that the used solvents do not reduce I Eu(III) ions, and sometimes they increase I by 16-45 %. 

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. 

The curves show that the peak capacity increases with the column efficiency, which is much as one would expect, however the major factor that influences peak capacity is clearly the capacity ratio of the last eluted peak. It follows that any aspect of the chromatographic system that might limit the value of (k ) for the last peak will also limit the peak capacity. Davis and Giddings [15] have pointed out that the theoretical peak capacity is an exaggerated value of the true peak capacity. They claim that the individual (k ) values for each solute in a realistic multi-component mixture will have a statistically irregular distribution. As they very adroitly point out, the solutes in a real sample do not array themselves conveniently along the chromatogram four standard deviations apart to provide the maximum peak capacity. 

A precise mastery of the chromatographic process also requires that the relative humidity be controlled. There are sufficient examples demonstrating that reproducible development is only possible if temperature and relative humidity are maintained constant. The influence of the latter on chromatographic behavior can be investigated using the Vario KS chamber (Fig. 59). When the relative humidity IS altered it is possible that not only the zone behavior will be changed but also the order of the zones on the chromatogram (Fig. 60). 

In theory, SEC of proteins depends only on their molecular size. Sometimes the size of a protein varies with the ionic strength of the buffer (5,6). The concentration of salt not only affects the conformation of the protein, but can also influence the chromatographic separation itself. Additional retention... 

Because temperature shifts may also influence the packing quality, the temperature should not be changed during the chromatographic step and the packing of the column should be done at the operation temperature. To prevent the denaturation of sensitive proteins, the chromatography is carried out in a cold chamber (or cabinet). For this purpose the column packing has to be performed at the same ambient temperature (store the gel before use at the same temperature ). 

Quantitative analysis using the internal standard method. The height and area of chromatographic peaks are affected not only by the amount of sample but also by fluctuations of the carrier gas flow rate, the column and detector temperatures, etc., i.e. by variations of those factors which influence the sensitivity and response of the detector. The effect of such variations can be eliminated by use of the internal standard method in which a known amount of a reference substance is added to the sample to be analysed before injection into the column. The requirements for an effective internal standard (Section 4.5) may be summarised as follows ... 

In these studies, choose different sets of affinities (SiB) and (S2B), and run these with the same parameters for the other ingredient encounters, as in Example 6.5. The cellular automata modeling of chromatographic separation produces a very realistic picture of the events taking place. It provides a visual and a tabular representation of the influence of variables on the process. The student is challenged to pursue these models and to compare them with some of the mathematical descriptions possible from chromatography. 

The chromatographic behaviour of condensed phosphates was found to be strongly influenced by water content and pH. The Ry of tris(dimethyl-amino)phosphine chalcogenides is generally lower than those of the trialkyl-phosphine chalcogenides but the difference is greatest for the P " compounds, and whereas the trialkylphosphines have the highest Ry values in the whole series, tris(dimethylamino)phosphine has the lowest. ... 

The activity tests of the catalyst were carried out in a microflow reactor set-up in which all the high temperature parts are constructed of hastelloy-C and monel. The reactor effluent was analyzed by an on-line gas chromatograph with an Ultimetal Q column (75 m x 0.53 mm), a flame ionization detector, and a thermal conductivity detector. The composition of the feed to the reactor can be varied, besides the temperature, pressure, and space velocity. The influence of the recycle components CHCIF2 and methane was tested by adding these components to the feed. In total five stability experiments of over 1600 hours were performed. In each... 

The column packing proced ire strongly influences the retention of large sized particles. The percentage of unretained particles of any size can be estimated from the ratio of its chromatogram areas obtained with and without the columns connected to the chromatograph. 

The reactor was charged with 37 elastomer which was Insoluble In the chromatographic mobile phase. Thus, the relationship between calculated and measured solids contents should have, and did, differ by at least 37 at low conversions. Expected formation of Insoluble graft polymer would also have Influenced relationship between calculated and measured total solids. 

For the same adsorbent, different mobile phases can be used according to the aim of chromatographic analysis. Rather than preparing an endless line of chromatographic plates of different thickness, it is easier to change the mobile phase up to the most convenient composition, keeping the same characteristics of the stationary phase. In the case of a hygroscopic adsorbent, the adsorbed water influences its activity. 

One of the most crucial influencing factors in planar chromatography is the vapor space and the interactions involved. The fact that the gas phase is present, in addition to stationary and mobile phases, makes planar chromatography different from other chromatographic techniques. Owing to the characteristic of an open system the stationary, mobile, and vapor phases interact with each other until they all are in equihbrium. This equilibrium is much faster obtained if chamber saturation is employed. This is the reason for differences in separation quality when saturated and unsaturated chambers are used. However, the humidity of the ambient air can also influence the activity of the layer and, thus, separation. Especially during sample application, the equihbrium between layer activity and relative humidity of the... 

Zarzycki and coworkers [77] studied the influence of temperature on the separation of cholesterol and bile acids using reversed-phase stationary phases. The best chromatographic conditions for the separation of mnlticomponent samples of steroids were chosen. Experiments were performed on wettable plates with RP-18W and at the temperatnres of 5, 10, 20, 30, 40, 50, and 60°C. The studies showed (Figure 9.9) that the degree of separation in the high-temperature region can be increased by an improvement of the efficiency of the chromatographic system. However, a relatively weak retention-temperatnre response for the studied steroids was observed. 

A particular problem with GRAFA and RBL is the reproducibility of the retention data. The retention time axes should be perfectly synchronized. Small shifts of one time interval (thus the ith spectrum in X, corresponds with the i+lth spectrum in X ) already introduce major errors (> 5%) when the chromatographic resolution is less than 0.6. The results of an extensive study on the influence of these factors on the accuracy of the results obtained by GRAFA and RBL have been reported in Ref. [37]. Although some practical applications have been reported [38,39], the lack of robustness of RBL and GRAFA due to artifacts mentioned above has limited their widespread application in chromatography. 

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