Reference standards characterization techniques

In thip appendix, a summary of the error propagation equations and objective functions used for standard characterization techniques are presented. These equations are Important for the evaluation of the errors associated with static measurements on the whole polymers and for the subsequent statistical comparison with the SEC estimates (see references 26 and 2J for a more detailed discussion of the equations). Among the models most widely used to correlate measured variables and polymer properties is the truncated power series model... 

An examination of the literature,10,21-24 authoritative guidances,6-9 and current industrial best practices, suggests that the analytical techniques in Table 1 be considered for the characterization of reference standards. Other techniques are occasionally employed but are not discussed here. These may include particle size analysis,25 nephelometry, heavy metals analysis,26 surface area,27 bulk density,28 pH,29 dissociation constants, microbiological testing30 and other spectroscopic measurements (e.g., NIR, fluorescence, CD, etc.). 

Ultraviolet-Visible Spectroscopy Ultraviolet-visible (UV-VIS) molecular absorption spectrophotometry (often called light absorption spectrophotometry or just UV-visible spectrophotometry) is a technique based on measuring the absorption of near-UV or visible radiation (180-770 nm) by molecules in solution.35,36 Reference standard characterization by UV-VIS spectophotometry includes determining the absorption spectra and the molar extinction coefficient. These two spectral characterizations are used as identifiers of reference standards. 

The characterization of ceramic thick films requires the use of special techniques in addition to those normally used to characterize bulk ceramics. This chapter has focused on these special techniques. The reader is referred to the following chapter for more information on the standard characterization techniques for ceramics, since many of them are also applicable to thick films. 

The /3-polymorphic form of anhydrous carbamazepine is official in the USP [3], The USP stipulates that, The X-ray diffraction pattern conforms to that of USP Carbamazepine Reference Standard, similarly determined. No limits have been set in the USP for the other polymorphs of anhydrous carbamazepine. Although several polymorphic forms of anhydrous carbamazepine have been reported, only the a- and /3-forms have been extensively studied and characterized [49]. A comparison of the powder x-ray diffraction patterns of these two forms revealed that the 10.1 A line (peak at 8.80° 26) was unique to a-carbamazepine, and so this line was used for the analysis (Fig. 5). It was possible to detect a-carbamazepine in a mixture where the weight fraction of a-carbamazepine was 0.02 at a signal-to-noise ratio of 2. Much greater sensitivity of this technique has been achieved in other systems. While studying the polymorphism of l,2-dihydro-6-neopentyl-2-oxonicotinic acid, Chao and Vail [50] used x-ray diffractometry to quantify form I in mixtures of forms I and II. They estimated that form I levels as low as 0.5% w/w can be determined by this technique. Similarly the a-inosine content in a mixture consisting of a- and /3-inosine was achieved with a detection limit of 0.4% w/w for a-inosine [51]. 

Further analysis is based on the idea that the characteristic experimental behavior of different classes of compounds and the suitability of those or other models used to describe this behavior is ultimately related to the extent to which the chromophores or electron groups physically present in the molecular system are reflected in these models. It is easy to notice, that the MM methods work well in case of molecules with local bonds designated in Table 1 as valence bonds the QC methods apply both to the valence bonded systems, and for the systems with delocalized bonds (referred as orbital bonds in Table 1). The TMCs of interest, however, not covered either by MM or by standard QC techniques can be physically characterized as those bearing the d-shell chromophore. The magnetic and optical properties characteristic for TMCs are related to d- or /-states of metal ions. The basic features in the electronic structure of TMCs of interest, distinguishing these compounds from others are the following ... 

While many of the standard electroanalytical techniques utilized with metal electrodes can be employed to characterize the semiconductor-electrolyte interface, one must be careful not to interpret the semiconductor response in terms of the standard diagnostics employed with metal electrodes. Fundamental to our understanding of the metal-electrolyte interface is the assumption that all potential applied to the back side of a metal electrode will appear at the metal electrode surface. That is, in the case of a metal electrode, a potential drop only appears on the solution side of the interface (i.e., via the electrode double layer and the bulk electrolyte resistance). This is not the case when a semiconductor is employed. If the semiconductor responds in an ideal manner, the potential applied to the back side of the electrode will be dropped across the internal electrode-electrolyte interface. This has two implications (1) the potential applied to a semiconducting electrode does not control the electrochemistry, and (2) in most cases there exists a built-in barrier to charge transfer at the semiconductor-electrolyte interface, so that, electrochemical reversible behavior can never exist. In order to understand the radically different response of a semiconductor to an applied external potential, one must explore the solid-state band structure of the semiconductor. This topic is treated at an introductory level in References 1 and 2. A more complete discussion can be found in References 3, 4, 5, and 6, along with a detailed review of the photoelectrochemical response of a wide variety of inorganic semiconducting materials. 

Table I lists the major characterization techniques which have been applied to the molybdena catalyst. They may be grouped into two broad categories nonspectroscopic and spectroscopic methods. Space does not permit a full discussion of the theory, experimental techniques, or interpretation of results of these techniques—we give here only the author s interpretations of their results. The reader is referred to any number of standard texts or reviews on the specific technique for a more complete description.

Once the structural features of a reference standard of the desired protein have been well characterized, lot-to-lot confirmation of identity can be conducted using a carefully selected group of tests, wherein the lot undergoing analysis is compared to the reference standard. Tests commonly employed for this purpose are listed in Table III. Peptide mapping is perhaps the most powerful and universally used technique since it provides relatively specific confirmation of correct primary sequence and, when non-reducing conditions are employed, can be used to confirm correct disulfide bond formation. Tertiary structure is difficult to address directly on a routine (lot-to-lot) basis, and the presence of correct biological activity is often used as evidence that the correct tertiary structure is maintained. 

Obviously, the universal goal of any measurement technique is to obtain reproducible results regardless whether the samples come from different sources with different matrix effects, are run by different operators, in different laboratories, on different occasions and using different lots of reagents. This is usually accomplished by characterization of the newly developed assay in terms of sensitivity, selectivity, robustness and correctness (i.e., accuracy and precision). Evaluation of sensitivity and precision does not normally constitute a problem for a newly developed immunoassay, and accuracy can be attained by comparison with the results obtained by a reference ( standard ) method. However, the evolution of assay standardization from this point on is much more difflcult. The following section deals with application-specific problems related to validation and standardization of immimoassay. 

The predominant mode of HPLC, reversed phase, involves the separation of material based on the partitioning between a relatively polar mobile phase and a nonpolar stationary phase. Normal phase HPLC—nonpolar mobile phase and polar stationary phase—is considered an orthogonal technique to reversed-phase HPLC when qualifying reference standards. In fact it is common for the elution order to be entirely reversed when switching an analysis from reversed to normal phase. Therefore, highly nonpolar impurities can be easily characterized by normal phase separations. 

Phase-solubility analysis17 (sometimes referred to as phase equilibrium purification) is the quantitative determination of the purity of a substance through the application of precise solubility measurements. At a given temperature, a definite amount of a pure substance is soluble in a definite quantity of solvent. The resulting solution is saturated with respect to the particular substance, but the solution remains unsaturated with respect to other substances even though such substances may be closely related in chemical structure and physical properties to the particular substance being tested. There are examples of the use of this technique in HPLC methods development18 and in the characterization of reference standards,19 but the... 

Once a well-characterized, pure reference standard of known concentration is available, one must be aware of the fact that the actual preparation of the assay calibrators can also influence the PK standard curve. The physical treatment and handling of concentrated protein solutions can be very challenging. Some reference standards require specific handling techniques to prevent aggregation or denaturation of the protein. The effects of vortexing can be disastrous as can the process used to thaw frozen reference standard or calibrator solutions. Details such as the dilution pattern that is used in spiking the calibration solutions can contribute to assay bias and between-laboratory discrepancies. 

In vitro assays do not use any whole-cell or animal-based components. The fibrin clot lysis assay, as established for tissue plasminogen activators and described for alteplase in the USP, is an example of this type of potency testing [5]. By means of defined standard materials, a fibrin clot is formed and the time to complete lysis is characterized as measure of potency, compared to a reference standard with defined activity. The LAL-test is a well-established and internationally harmonized in vitro alternative to detect or quantify bacterial endotoxins, using Limulus amebocyte lysate (LAL) obtained from the aqueous extracts of circulating amebocytes of horseshoe crab (Limulus polyphemus or Tachypleus tri-dentatus) which has been prepared and characterized appropriately [5]. Two types of technique may be used for this test gel-clot techniques, which are based on gel formation and photometric techniques. 

The peptide sequence in a protein can now be determined with automated equipment. A standard reference on this technique is the Handbook of Protein Sequence Analysis. However, many synthetic copolymers cannot be taken apart with such precision they must be analyzed as intact chains. Consider an AB copolymer. The composition can be specified by the mole fraction = 1 - Xg. While some synthetic copolymers have an identical sequence distribution for each chain, most chains are characterized by sequence probability distributions, One example of a unique sequence distribu-... 

The predorninant method for the analysis of alurninum-base alloys is spark source emission spectroscopy. SoHd metal samples are sparked direcdy, simultaneously eroding the metal surface, vaporizing the metal, and exciting the atomic vapor to emit light ia proportion to the amount of material present. Standard spark emission analytical techniques are described in ASTM ElOl, E607, E1251 and E716 (36). A wide variety of weU-characterized soHd reference materials are available from major aluminum producers for instmment caUbration. 

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