Retention time problems

We divided this section into two major subsections peak-shape problems and retention-time problems. The last section then covers problems that do not fit into the first two categories, such as pressure problems. This structure should allow you to quickly move to the chapter that covers the problem that you are trying to solve. In the section of peak-shape problems, example chromatograms should also help to point you to the most appropriate section. While this structure is good for rapid troubleshooting, it also makes repetition unavoidable. Therefore, we would like to remind the reader that this part of the book is not designed to be read as a consecutive text. 

In this section, we will discuss various causes of retention-time problems and examine their causes. We will discuss the influence of temperature, mobile-phase composition, column contamination, column aging, and various other topics. We will also briefly discuss again column-to-column reproducibility. 

The chapter is divided into two sections. In the first section we deal with retention time problems that are random in nature. The second section then covers the cases where the retention times are drifting in one direction only. If we can differentiate between these cases, we can immediately exclude some causes of the observed problem. For example, random fluctuations of retention times are not a symptom of column aging. 

Using the data from Problem 1, calculate the resolution and selectivity factors for each pair of adjacent compounds. For resolution, use both equations 12.1 and 12.21, and compare your results. Discuss how you might improve the resolution between compounds B and C. The retention time for an unretained solute is 1.19 min. 

Chiral separations present special problems for vaUdation. Typically, in the absence of spectroscopic confirmation (eg, mass spectral or infrared data), conventional separations are vaUdated by analysing "pure" samples under identical chromatographic conditions. Often, two or more chromatographic stationary phases, which are known to interact with the analyte through different retention mechanisms, are used. If the pure sample and the unknown have identical retention times under each set of conditions, the identity of the unknown is assumed to be the same as the pure sample. However, often the chiral separation that is obtained with one type of column may not be achievable with any other type of chiral stationary phase. In addition, "pure" enantiomers are generally not available. 

A discussion of retention time in rotary Idlns is given in Brit. Chem. Eng., 27-29 (Januaiy 1966). Rotary-ldln heat control is discussed in detail by Bauer [Chem. Eng., 193-200 (May 1954)] and Zubrzycki [Chem. Can., 33-37 (Februaiy 1957)]. Reduction of iron ore in rotaiy Idlns is described by Stewart [Min. Congr J., 34—38 (December 1958)]. The use of balls to improve solids flow is discussed in [Chem. Eng., 120-222 (March 1956)]. Brisbane examined problems of shell deformation [ Min. Eng., 210-212 (Februaiy 1956)]. Instrumentation is discussed by Dixon [Ind. Eng. Chem. Process Des. Dev., 1436-1441 (July 1954)], and a mathematical simulation of a rotaiy Idln was developed by Sass [Ind. Eng. Chem. Process Des. Dev., 532-535 (October 1967)]. This last paper employed the empirical convection heat-transfer coefficient given previously, and its use is discussed in later correspondence [ibid., 318-319 (April 1968)]. 

If the mobile phase is a liquid, and can be considered incompressible, then the volume of the mobile phase eluted from the column, between the injection and the peak maximum, can be easily obtained from the product of the flow rate and the retention time. For more precise measurements, the volume of eluent can be directly measured volumetrically by means of a burette or other suitable volume measuring vessel that is placed at the end of the column. If the mobile phase is compressible, however, the volume of mobile phase that passes through the column, measured at the exit, will no longer represent the true retention volume, as the volume flow will increase continuously along the column as the pressure falls. This problem was solved by James and Martin [3], who derived a correction factor that allowed the actual retention volume to be calculated from the retention volume measured at the column outlet at atmospheric pressure, and a function of the inlet/outlet pressure ratio. This correction factor can be derived as follows. 

Where the retention time is actually specified can present a problem. If you specify it in a general procedure you are likely to want to prescribe a single figure, say five years for all records. However, this may cause storage problems - it may be more appropriate, therefore, to specify the retention times in the procedures that describe the records. In this way you can be selective. 

A more difficult criterion to meet with flow markers is that the polymer samples not contain interferents that coelute with or very near the flow marker and either affect its retention time or the ability of the analyst to reproducibly identify the retention time of the peak. Water is a ubiquitous problem in nonaqueous GPC and, when using a refractive index detector, it can cause a variable magnitude, negative area peak that may coelute with certain choices of totally permeated flow markers. This variable area negative peak may alter the apparent position of the flow marker when the flow rate has actually been invariant, thereby causing the user to falsely adjust data to compensate for the flow error. Similar problems can occur with the elution of positive peaks that are not exactly identical in elution to the totally permeated flow marker. Species that often contribute to these problems are residual monomer, reactants, surfactants, by-products, or buffers from the synthesis of the polymer. 

Chlorambucil - there is no problem with the quantitation ion (at m/z 254), although the second ion proves to be a little difficult. While the ion at m/z 303 is the obvious choice, this is not very intense and therefore for samples containing small amounts of analyte the precision of measurement of this ion will be reduced and it may not be detectable at all levels at which the quantitation ion is observed. We could possibly consider the (M- -2) ion, as the combination o/m/z 254 (high mass, and therefore reasonable specificity), the presence of one chlorine, and the chromatographic retention time could be considered sufficient for definitive identification in those cases in which the intensity o/m/z 303 is insufficient. 

In the fluidized bed process, attrition caused dry sorbent to be carryover. This mainly occurred in the early stage of fluidization and was highly affected by gas velocity. The amount of attrition of molecular sieve 5 A and molecular sieve 13X were larger than those of activated carbon and activated alumina. In addition, percentage losses of adsorption capacities of molecular sieve 5A and molecular 13X were 14.5% and 13.5%, whereas those of activated carbon and activated alumina were 8.3% and 8.1%, respectively. This is because retention time of molecular sieve 5A and molecular 13X decreased due to elutriation of particle generated from attrition. Also, Ka of activated alumina and activated carbon were the lower than those of Molecular sieve 13X and 5A. Consequently, molecular sieve 5A and molecular 13X could cause high maintenance cost for dry sorbent and problems in the operation of fluidized bed process. 

In analysis of homopolymers the critical interpretation problems are calibration of retention time for molecular weight and allowance for the imperfect re >lution of the GPC. In copolymer analysis these interpretation problems remain but are ven added dimensions by the simultaneous presence of molecular weight distribution, copolymer composition distribution and monomer sequence length distribution. Since, the GPC usu y separates on the basis of "molecular size" in solution and not on the basB of any one of these particular properties, this means that at any retention time there can be distributions of all three. The usual GPC chromatogram then represents a r onse to the concentration of some avera of e h of these properties at each retention time. 

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. 

Residual silanol groups in chemically bonded phases have been associated with a number of undesirable interactions with polar solutes such as excessive peak tailing, irreproducible retention times, and excessively long retention times. These problems are particularly prevalent for amines and other strong bases. A large number of test systems have been proposed to characterize the concentration of residual silanol groups on bonded phase packings, and some representative examples are... 

Figure 3.6 Different peak distortion problems due to band broadening in time and band broadening in space observed during hot splitless injection. Band broadening in space is characterized by a broadening which grows proportionally with retention time and may result in peak splitting that is poorly reproducible. Band broadening in time is characterized by a constant broadening of all peaks. Partial solvent trapping results in characteristic chair and stool shaped peaks. (Adapted with permission from ref.

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