Calibration of retention

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. 

Quantifications are by internal standardization with azobenzene after calibration of retentions (see Table 2) and nitrogen-phosphorus detector (NPD) response factors for authentic compounds. Response factors are determined by these steps ... 

Quantification of nitroso-alkaloids is by internal standardization. Calibration of retention times and response factors is made with authentic nitrosamines. 

Carr and Schelling6 state in detail how the reservations many chromatographers still have toward gradient elution no longer hold true ...gradient elution provides an overall faster analysis, narrower peaks and similar resolution of the critical pair compared to isocratic elution without loss in repeatability of retention time, peak area, peak height, or linearity of the calibration curve. ... 

Unfortunately, none of the commonly used molecular probes is adequate to evaluate column-to-column variabilities [88]. The absolute prediction of retention of any compound involves the use of a rather complex equation [89,90] that necessitates the knowledge of various parameters for both the solute and the solvent [91]. The relative prediction of retention is based on the existence of a calibration line describing the linearity between log and interaction index. This second approach, although less general than the first, is simpler to use in practice, and it often gives more accurate results than the first. With a proper choice of calibration solutes, it is possible to take into account subtle mobile phase effects that cannot be included in the theoretical treatment. 

For calibration, the solutions were prepared overnight at ambiant temperature without agitation, filtration or addition of antioxidants (a mild agitation and filtration resulted in an increase of retention time, RT, by 0.40 min, equivalent to a reduction of molecular weight by 26%). The calibration curve for the four columns Figure 1 was non-linear addition of the fifth, 500 A column. Figure 2 linearized the dependence ... 

Figure 4. Calibration of GPC retention voliime using known compounds.

The GPC of a local crude (Bryan, Texas) sample spiked with a known mixture of n-alkanes and aromatics is shown in Figure 5 and the GPC of the crude is shown in Figure 6. The hydrocarbon mixture is used to calibrate the length of the species which separates as a function of retention volume. Ttie molecular length is expressed as n-alkane carboa units although n-alkanes represent only a fraction of the hydrocarbons in the crude. In addition to n-alkanes, petroleum crude is composed of major classes of hydrocarbons such as branched and cyclic alkanes, branched and cyclic olefins and various aromatics and nonvolatiles namely asphaltenes. Almost all of the known aromatics without side chains elute after n-hexane (Cg). If the aromatics have long side chains, the linear molecular size increases and the retention volume is reduced. Cyclic alkanes have retention volumes similar to those of aromatics. GPC separates crude on the basis of linear molecular size and the species are spread over 10 to 20 ml retention volume range and almost all of the species are smaller than the polystyrene standard (37A). In other words, the crude has very little asphaltenes. The linear... 

Calibration of size-exclusion chromatography columns based on the finding that the retention volume of a molecular or particulate species is usually a single-valued function of an appropriate size parameter of this molecule or particle, irrespective of its chemical nature and structure. 

When the total polymer response, is known as a function of retention volume, the molecular weight distributlon can be obtained in the usual manner with the appropriate molecular weight calibration curve. The molecular weight calibration curve can be obtained (a) by using the Runyon (65) copolymer molecular weight scale approach, or (b) by using a hydrodynamic volume approach if the Mark-Houwink constants for the polymer of interest are known or can be determined, or (c) by using a hydrodynamic volume approach in conjunction with an on-line viscosity detector. 

Does the system include the retention of calibration certificates or data used in support of all calibration of measuring equipment Does the record system allow for calling forward, at the appropriate interval, equipment requiring calibration ... 

Yau et al.f used polystyrene samples of known molecular weight in tetrahydrofuran to calibrate the retention volume of a size-exclusion chromatograph. They obtained the following results ... 

An equation can be derived relating Z A (dtFE) to Z gF ( i>thf ) As was pointed out in step C, the retention volume calibration curve relating i>thf to i>tfe was constructed by relating t>rFE to i>thf at points of equal weight percent polymer on the integral distribution of retention volume curves in tetrahydrofuran and in TFE. At these points the molecular weight of the polymer species in tetrahydrofuran is the same as the molecular weight of the polymer species in TFE. 

Note Procedural controls for computer applications may include development, use, and support of systems, data handling, storage and retrieval, maintenance, repair, calibration of equipment, management and personnel responsibilities, record retention, training. 

Compound identification in GC/MS methods is based on the retention time and the mass spectra interpretation the quantitation is based on the abundance of a primary (characteristic) ion. For a compound to be positively identified, all of the ions in the spectrum must be detected at one and the same retention time, which corresponds to the retention time of this compound in the calibration standard. The retention times of the primary ion and one or two secondary ions are typically monitored for this purpose. A combination of retention time and mass spectra is a formidable compound identification tool. That is why compound identification mistakes are relatively rare in GC/MS analyses, and they usually occur due to analysts lack of experience. 

The performance of the system is tested by injecting 2 xl of OPCW check mixture running the Performance Check Injection method. The composition of the mixture is given in Annex 2. The retention times of the series of nine hydrocarbons in the check mixture are used by AMDIS for calibration of the RI the other seven compounds are used for assessing the performance of the GC and MS part of the system. Two components of the check mixture, chloromethylaniline and dibenzothiophene, are used for evaluation of isotopic ratios for chlorine and sulfur measured by the mass spectrometer. The example chromatogram of the check mixture is presented in Figure 5. 

In most cases, absolute retention times are used for the identification. Modem GCs have high precision and accuracy of retention times within 0.05 %, or better can be obtained in subsequent runs during the same day. When the analysis is repeated by another instrument, the deviation may be of several percent. The reproducibility of absolute retention times is strongly dependent on the proper adjustment of all chromatographic parameters. In addition, the column properties are not exactly the same even when similar columns from the same manufacturer are used. On the other hand, absolute retention times are the most useful identification parameters if a few chemicals are to be monitored and the background is low. This method requires frequent calibration because even small changes in chromatographic conditions will influence the absolute retention times. 

The capacity value (retention time - column dead time) is used for the calculation of the log D. For this purpose a standard set of compounds with known log D7.4 is used for calibration of the system. 

The variety of stationary phases available commercially and the lack of standardisation between different laboratories makes it difficult to compare the retention factors k directly as comparison of lipophilicity. Therefore different lipophilicity scales obtained from chromatographic data (isocratic or gradient elution) have been introduced. A calibration of the individual chromatographic system with compounds of known lipophilicity is used in all cases. 

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