Column reproducibilities

The theoretical values were confirmed by testing commercial columns. [Reproduced from Hagel (1992) with permission.]... 

Figure 1.5 Representation of a coupled column system consisting of a primary column and two secondary columns (reproduced with permission from reference (30)).

Figure 3.7 [continued) (b) Chromatograms of (iii) the dichloromethane extract of strawberry fruit yoghurt analysed with an apolar primary column, with the heart-cut regions indicated, and (iv) a non-racemic mixture of y-deca-(Cio) and 7-dodeca-Cj2 lactones isolated by heart-cut transfer, and separated by using a chiral selective modified cyclodextrin column. Reproduced from A. Mosandl, et al J. High Resol. Chromatogr. 1989, 12, 532 (39f. 

Figure 3.8 Second-dimension chiral cyclodextrin capillary column separation of a non-racemic pair of nonachlorobomane compounds extracted from dolphin blubber, shown with expanded attenuation in the inset. The primary separation (not shown) was performed on an apolar primary capillary column. Reproduced from H.-J. de Geus et al. J. High Resol. Chromatogr. 1998, 21, 39 (59).

Figure 8.20 Scheaatic representation of process of (A) pairtial concurrent evaporation and (B) concurrent evaporation using a loop-type interface for the transfer of eluent fron an VC colunn to a 6C column. (Reproduced with permission from ref. 224. Copyright Elsevier Scientific Publishers).

Figure 4.37 Preparative-scale separation of bilirubin laomarm by high pressure liquid chronatography. The analytical separation was optimized to maximize the separation factor at the smallest practical value for the capacity factor and then the saiq>le size scaled-up to that allowed by the larger amount of packing in the preparative column. (Reproduced with permission from Perkin-Elmer Corporation).

Figure 5.2 A, valve, column and flow cell asseidily of a ainiaturized liquid chromatograph for use with small bore columns (Reproduced with permission from ref. 14) and B, mobile phase reservoir designed for solvent degassing by heat and helium sparging (Reproduced with permission from ref. 34. Copyright Elsevier Scientific Publishing Co.)...

Figure 8.16 A simple two-dimensional gas chrtwatograph for packed column fractionation or enrichment using a Deans switch and intermediate trap for transfer to a capillary column. (Reproduced with permission from ref. 205. Copyright Dr. Alfred Huethig Publishers).

Figure 8.18 Schenatic diagram of a dual oven two-dimensional gas chromatograph employing live switching between two capillary columns. (Reproduced with permission from Siemens AG).

Before releasing a process column for chromatography, it is advisable to perform some test to measure efficiency, such as calculating height equivalent theoretical plates (HETP), both to forestall any problems in the column bed and to provide a benchmark by which to measure column reproducibility and predict degradation of the bed or material. Examples of compounds that are relatively innocuous for use in pharmaceutical applications are 1% NaCl (for gel filtration), concentrated buffer solutions (for ion exchange), and benzyl alcohol and parabens for reverse phase LC.10... 

The determination of precision can divided into three categories, namely repeatability, intermediate precision, and reproducibility. Repeatability, or intraassay within-day precision, is determined when the analysis is performed in one laboratory by one analyst, using one definite piece of equipment, and is performed within one working day. Intermediate precision is obtained when the analysis is performed within a single laboratory by different analysts over a number days or weeks, using different equipment, reagents, and columns. Reproducibility represents the... 

MINIMIZING VARIATION IN THE ANALYTICAL SYSTEM Column Reproducibility... 

FIGURE 9 Scanning electron micrograph of monolithic silica-based capillary column. (Reproduced with permission from reference 55.)... 

The application of HPLC in routine environments, like pharmaceutical, food, or environmental analysis and particularly quality assurance, makes not only great demands on the robnstness of HPLC hardware, comprising pumps, column thermostats, and detection units, bnt in addition to the column reproducibility. Column reproducibility can be investigated at different levels of complexity Run-to-run reproducibility compares consecutive chromatographic runs, whereas long-term stability describes the column variance over several hundreds of injections. Column-to-column (batch-to-batch) reproducibility finally explores the match of independently fabricated chromatographic columns. Column characteristics that are routinely consulted for the determination of the robustness are retention, selectivity, column efficiency, and peak symmetry. 

Figure 2.9—Gas analysis. On the left is one of the first chromatograms ever obtained, point by point, representing a mixture of air, ethylene and acetylene separated on silica gel (E. Cremer and F. Prior, Z. Elektrochem. 1951, 55, 66). On the right is a gas analysis obtained on a PLOT column (reproduced by permission of Supelco).

Figure 20.4—Static mode of headspace sample analysis. The sampling phial is pressurised with the carrier gas of the chromatograph. After equilibrium, a small volume of the gas containing the volatile compounds is inserted into a sample loop. Rotation of the six-way valve allows introduction of the sample into the injector of the chromatograph. Consequently, this set-up combines sample preparation with sample introduction into the chromatographic column. (Reproduced by permission of Tekmar.)...

Relevant parameters for column-to-column reproducibility data are plate number, peak symmetry, selectivity, and adsorption phenomena checked with chromatography of amines and acids. For column life, the analyst must periodically check k loss, change in asymmetry factor, and plate number. 

Liquid chromatography is now a mature technique. Instruments are reliable and increasingly computer assisted. Column-to-column reproducibility is ensured by most manufacturers. The quest for the universal detector is about to end with the advent of a sophisticated and miniaturized MS detector. Development of a method can be achieved in a rather short period with available software. The emphasis is on validation more than on how to handle it. Capillary columns are sure to improve, and the trend will be toward many parallel analyses. 

Fig. 19. Binding constants observed for the complexation of diamines by the bis-zincporphyrin peptide 58 (striped columns) and a mono-zincporphyrin tripeptide analog (BOC-A-Zn-porphynn-E-A-OMe) (solid columns). (Reproduced with the permission of Ref. 1)...

Figure4.22 Separation of oligodeoxy thymidylate fragments containing 12-18 nucleotide sub-units (dT12 18) on calixpyrrole modified silica gel column. (Reproduced by permission of The Royal Society of Chemistry).

Structure Macroporous polystyrene-divinylbenzene nonpolar adsorbent, 62-177 pm particle size Analytical Properties Used mainly in preparative-scale HPLC stable over entire pH range (1-13) sometimes difficult to achieve column-to-column reproducibility due to packing the irregular particles relatively lower efficiency than alkyl bonded phases particles tend to swell as the organic content of the mobile phase increases Reference 1... 

Tests of the reproducibility of retention times, retention factors, separation selec-tivities, and column efficiencies for our methacrylate monolithic capillary columns are summarized in Table 6.2. This table shows averaged data obtained for 9 different analytes injected 14 times repeatedly every other day over a period of 6 days, as well as for 7 different capillary columns prepared from the same polymerization mixture. As expected, both injection-to-injection and day-to-day reproducibilities measured for the same column are very good. Slightly larger RSD values were observed for col-umn-to-column reproducibility. While the selectivity effectively did not change, larger differences were found for the efficiencies of the columns. 

For the evaluation of any separation medium, three types of characterization are important in determining the overall usefulness of the material within column reproducibility, column-to-column reproducibility and stability. All of these factors have been tested for the etched chemically modified capillaries in order to determine the reproducibility of the etching and chemical modification processes as well as the ruggedness of the silanization/hydrosilation method for the attachment of various organic moieties to the roughened inner wall. 

Several types of etched capillaries have been tested with respect to within column reproducibility. For example, the reproducibility of the migration times for 151 consecutive injections of the proteins lysozyme and ribonuclease A was tested on an etched Ci8 modified column at pH = 3.0 [20]. It was found that both solutes gave no discernible increase or decrease in migration time (tM) and its overall reproducibility... 

While retentivity specifications are useful, an even better and more sensitive test for column-to-column reproducibility is the selectivity factor, alpha. This factor measures the ratio of the retentivity or capacity factors (k ) of two solutes and determines if there are any differences between columns which would result in the two peaks moving closer together or further apart. 

---