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example. However, molecules with large axial ratios (i.e. rod-like in shape rather than globular; e.g. collagen) behave as if they have a much greater mass on gel ltration. Where little is known about the overall shape of the protein, care should be taken in interpreting gel ltration data. A further point to note is that some proteins may experience secondary ionic or ion exchange interactions with the stationary phase beads. This can result in the molecule being retained on gel ltration in a way that may not depend on mass alone. One means of avoiding such effects is to carry out the experiment in the presence of 100 300 mM NaCl or a similar salt. The mass of individual polypeptides or subunits of which oligomeric proteins are composed may be determined under denaturing conditions by a number of techniques such as SDS polyacrylamide gel electrophoresis ( 5) or mass spectrometry ( 4). Determination of native mass may help us to determine the quaternary structure of proteins by comparison with results from denaturing experiments. A third application of gel ltration chromatography is in protein puri cation. Since the method depends on molecular mass, it may be used to separate proteins based on differences in their mass. An important limitation to this approach is the loading volume that can be achieved. Generally, this is a maximum of approximately 5% of the bed volume of the column. This technique is therefore often used near the end of a puri cation strategy as a polishing step. Neither gel ltration nor ion exchange chromatography alone is capable of achieving complete puri cation of proteins. However, since it is unlikely that two proteins will have the same net charge and mass, combining the two approaches can often achieve impressive puri cation (Section 2.10.1). 2.4.3 Reversed Phase



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This shows that the log-likelihood function induced by p ( t ) is maximized by p = p ( i ) . In this sense, p ( 0 is the maximum likelihood estimator of p(8). H





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Extent of Validation Depends on Type of Method On the one hand, the extent of validation and the choice of performance parameters to be evaluated depend on the status and experience of the analytical method On the other hand, the validation plan is determined by the analytical requirement(s) as de ned on the basis of customer needs or as laid down in regulations When the method has been fully validated according to an international protocol [63, 68] before, the laboratory does not need to conduct extensive in-house validation studies It must only verify that it can achieve the same performance characteristics as outlined in the collaborative study As a minimum, precision, bias, linearity, and ruggedness studies should be undertaken.

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As expected, DABCO is a much stronger base than triethylamine, has two ionization potentials separated by several electron-volts, and is more easily oxidized than trimethylamine What is unusual about DABCO is that the in-phase combination of the nonbonded pairs is the HOMO, the lowest IP is lower than that of trimethylamine, and the next higher IP is higher than that of trimethylamine Explain Answer This question asks us to explain some unexpected features of a fairly simple molecule Based on the simplest application of orbital interaction theory, as shown in Figure B37a, one would expect the HOMO and HOMO-1 of DABCO to be the HH H out-of-phase a2 and in-phase a1 combinations of the interacting nitrogen lone pairs, respectively.

The reference point may be taken to be the energy of one of the N lone pairs, nN , which can also be taken as the energy of the HOMO of trimethylamine, that is, the IP by Koopmans' theorem Each nitrogen atom is a pyramidal tricoordinated atom, and the single remaining valence orbital is an sp n hybrid directed away from the point of the pyramid The two interacting sp n hybrid orbitals are pointed away from each other and so will interact only weakly through the smaller back lobes Nevertheless, one would expect to observe that DABCO has one IP which is lower than that of trimethylamine (ionization from the HOMO) and one which is higher (ionization from the HOMO-1).

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