Refractive index detectors universal calibration

Other analysis methods dependent on multiple detectors can be implemented using this automated system. Two methods under development are the use of a continuous viscometer detector with a refractive index detector to yield absolute molecular weight and branching, utilizing the universal calibration curve concept (4), and the use of a UV or IR detector with the refractive index detector to measure compositional distribution as a function of molecular weight. 

Absolute MWD can be measured using light scattering or viscometry combined with universal calibration. Compositional drift over the MWD of a polymer can be measured using a UV spectrophotometer and a differential refractive index detector. The increase in the available information also expands the complexity of data analysis. We discuss some of the concerns regarding data analysis that arise in multidetector SEC. 

The most commonly used detector is the refractive index detector (cf. Section 3.11.3), considered as universal since for polymers a variation of the refractive index is at first approximation independent of the molecular mass. As this detector is not very sensitive, other detectors are sometimes added to it. They are based upon the light absorption (UV detector) or fluorescence or light scattering (Figure 7.4). This last detector provides a more uniform response to structurally similar analytes than do light absorbtion detectors. Users can create a universal calibration set from a single analyte to quantify all analytes of the same class. 

When an on-line viscometer is used together with the refractive index detector to generate the intrinsic viscosity [t]] in order to build the universal calibration curve. Sec. II.B, the intrinsic viscosity [t]] can also be used to determine the presence and degree of branching. This is done by plotting the log of [t]] versus log molecular-weight for each slice of the distribution. This plot is called the viscosity law plot, or the Mark-Houwink plot. It is described by the equation... 

The combination of the differential refractive index (RI) detector and on-line viscometer allows the direct use of the universal calibration and thus true molecular weight determination. The RI detector is concentration-sensitive, and the viscometer records specific viscosity. The ratio of the specihc viscosity to the concentration is equivalent to intrinsic viscosity (as discussed in Section 6.1), and the continuous dependence of this ratio versus the retention volume could be related to the universal calibration curve, thus allowing the correlation of each point on the chromatogram with the true molecular weight. 

Cotton fibers are single cells composed primarily ( 96%) of the polymer cellulose. In our laboratory (5), cotton fibers were dissolved directly in the solvent DMAC-LiCl. This procedure solubilizes fiber cell wall components directly without prior extraction or derivatization, processes that could lead to degradation of high MW components. MW determinations have been carried out by a size-exclusion chromatography (SEC) system using commercial columns and instrumentation with DMAC-LiCl as the mobile phase. Incorporation of viscometry and refractive index (RI) detectors (6) allowed application of the universal calibration concept (7) to obtain MW distributions (MWDs) based on well-characterized narrow-distribution polystyrene standards (5). The universal calibration concept used by incorporation of dual detectors bypasses the need for cellulose standards. There are no cellulose standards available. Polystyrene standards for a wide range of MWs dissolved readily in DMAC-0.5% LiCl with no activation necessary. 

The application of dual detection [UV and refractive index (RI)] to the SEC analysis of polystyrene-poly(methyl methacrylate) (PS-PMMA) has already been studied in this laboratory (2). Both MWD and CCD were determined using a methodology outlined by Runyon et al. (3). This approach relies on SEC column calibration with narrow polydis-persity standards for each of the homopolymers as well as a measure of the detector response factors for each homopolymer to produce a copolymer MWD. In the case of PS and PMMA this is feasible, but in other block copolymer systems the availability of suitable molecular weight standards may be more limited. In addition, this procedure does rely on true SEC and is not valid for block copolymers for which the universal calibration does not hold true for both blocks in a given solvent system. 

To obtain the MMD, the mass of the polymer being eluted must be measured. This can be achieved continuously using refractive index (RI), UV, or IR detectors, which will give a mass distribution as a function of V. It is still necessary to estimate the molar mass of each fraction before the MMD curve can be constructed. If the universal calibration curve is valid for the system, then... 

SEC experiments were carried out on a Watters 150CV instrument (Waters Associates, Milford, Massachusetts, U.S.A.) equipped with both differential refractive index single-capUlary viscometer detectors. The solvent/mobile phase was H20/0.02% NaNs, at the flow rate of 1.0 ml/ min. Pump, solvent, and detector compartments were maintained at 50°C. Separation occurred over a column bank consisting of three analytical columns preceded by a guard column Shodex KB-G, KS-802, KS-803, and KB-804 (Phenomenex, Torrance, California, U.S.A.). Universal calibration was performed using a series of oligosaccharides (Sigma, St. Louis, Missouri, U.S.A.), and PuUuIan Standards (American Polymer Standards, Mentor, Ohio, U.S.A., and Polymer Laboratories, Amherst, Massachusetts, U.S.A.). 

In universal calibration, samples of a monodisperse polymer, often polystyrene, that is different from the polymer to be analyzed, are dissolved in the solvent of interest, and the intrinsic viscosities of the resulting solutions are measured. Then identical samples are injected into the column to be used, and the refractive index of the effluent is measured as a function of retention volume, V, which depends on V,. Then a calibration plot of [tj] M versus is prepared, and this plot is assumed to be valid also for the polymer to be analyzed. The retention time can also be used as the independent variable, since it is linear in at constant flow rate. It is convenient to fit an equation, for example a third-order polynomial, to the universal calibration curve. Carrying this concept a step further, if an on-line IV detector is used along with the DRI detector, the data from an analysis can be interpreted directly in terms of a molecular size distribution, and from this the MWD can be determined. 

A clever way to make a universal LC method more reliable for identification of surfactants in shampoos and hair conditioners was described by Kadano et al. (20). Two detectors were used, refractive index and UV. The ratio of the two responses was recorded versus retention time, giving a more specific signal than the output of either detector alone. Of course, calibration with the proper standards is all-important. Modern photodiode array UV detectors permit using a similar approach detection at two or more wavelengths, with automatic calculation of the ratio of the absorbance at different wavelengths. 

---