Other Chromatographic Methods

Capillary electrophoresis (CE) (see Section 3.5) has been used to determine partition coefficients [320-322]. Lipid vesicles or micelles are added to the buffer whose pH is adjusted to different values. Since drug molecules partition to a different extent as a function of pH, the analysis of mobility vs pH data yields log P values. 

Centrifugal partition chromatography (CPC) has been used to characterize the partitioning behavior of hydrophilic molecules, where log D values as low as —3 can be obtained [371,377-379]. It is not as popular a method as it used to be, apparently due to instrumental challenges. Cyclic voltammetry (CV) has become the new method used to get access to very low log D values, with partition coefficients reported as low as —9.8 [261,269,362]. 

If the two phases are equal in volume (1 1) and the substance is lipophilic, a very simple relationship can be applied to determine log P  

Note that for a weak acid, the octanol causes the Bjerrum curve to shift in the direction of higher pH, whereas for a weak base, octanol causes the shift to lower values of pH. Equation (4.14) may be applied to the molecules in Fig. 4.7, and logP deduced from the shifts in the curves. 

For diprotic molecules, 12 different characteristic shift patterns have been identified for cases where two species may partition simultaneously into the lipid phase [347]. Three of these cases are shown in Fig. 4.8, picking familiar drug substances as examples. Once the approximate constants are obtained from Bjerrum analysis, 

Monoclonal antibodies are inherently stable proteins. Being secretory products they are stabilized by disulfide bonds and not readily susceptible to damage by oxidation. In our experience, culture supernatants containing IgG antibodies remained active for at least 15 years at 4 °C when Hepes and sodium azide were 

Hydroxyapatite, Caio(P04)s(Ol[)2, is a unique substance which is both the reactive ligand and the matrix. It contains a combination of both positively charged (Ca ) and negatively charged binding sites which interact with 

There are so many derivatives of antibodies, proteolytic fragments, and genetically engineered variants that it is impossible to give specific recommendations for their purification however the general principles will still be applicable. 

Aggregation Size exciusion HPLC, native gel electrophoresis 

The concentration of a purified antibody is best measured by absorbance at 280 nm. For accurate results ensure that the spectrophotometer is properly cahbrated. The absorbance of a 1 mg/ml solution in a 1 cm cell is conventionally taken to be approx. 1.4 for IgG and 1.2 for IgM. The true values will vary from 

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Nova-Pak C18 column in a methanol water chloroform gradient.92 Choline chloride was added to the mobile phase. One review of techniques used in the analysis of triacylglycerols lists over 300 references on separations of the triglyceride fraction of fats using nonaqueous RPLC, aqueous RPLC, argen-tation chromatography, and other chromatographic methods.93... 

It is also becoming more common to combine IEC with other chromatographic methods to optimize glycoprotein characterization. Takahashi160 has developed a three-dimensional sugar mapping technique utilizing three different HPLC columns a DEAE column, a hydrophobic octadecylsilica... 

As with other chromatographic methods, there are a number of electrophoretic methods, including paper and gel electrophoreses and CE. Electrophoresis uses an electric current to move ionic species, either simple ions, amino acids, or complex proteins, through a medium (i.e., a gel) or a capillary (i.e., CE). During this process, typically, the ionic species move at different rates and are thus separated. 

In conjunction with densitometric detection, it can be used as a quantitative technique for compounds which are difficult to analyse by other chromatographic methods because of the absence of a chromophore. 

Polyst3rrene (Figure 4), which is a pol)rmeric support is also imsuitable in its original form for affinity separations due to the highly hydrophobic character. Native polyst3rrene, which is often used as a reversed-phase material, must be first rendered hydrophilic by one of various surface-coating techniques before used in other chromatographic methods [8]. 

The stationary phase in SEC is a highly porous substrate whose pores are penetrated best by small solute molecules. Because larger solute molecules are unable to enter as deeply into the pores, they will travel further down the column in the same time. The largest molecules, which are totally excluded from the pores, are eluted first from the column. Because the solvent molecules are usually the smallest, they are normally the last to be eluted. The rest of the solute molecules are eluted between these two extremes, at a time dependent on their ability to penetrate into the pores. In SEC, therefore, unlike other chromatographic methods, the entire sample often is eluted before the solvent dead time peak, t0, as shown in Figure 2.13.53... 

Among the chromatographic methods, ion exchange is the most commonly used in protein purification, due to its simplicity for scale-up, wide applicability, and low cost compared with other chromatographic methods. Ion exchange of proteins involves their adsorption onto the charged... 

Usually, the stationary phases for ion chromatography have a low capacity, in order to reduce the background signal in conductivity detection. Selectivity in ion chromatography can be optimized along the same lines as other chromatographic methods [368]. 

This chromatographic method usually produces very good separations of fiavonoids that are difficult to be resolved by other chromatographic methods. The main drawback of this method is the elevated cost of the stationary phase, preventingits use for large scale preparations, but it is quite useful for laboratory scale preparations. Mobile phases like... 

Beside HPLC, TLC is also commonly used in pharmaceutical analysis. Although it is less selective and less precise than other chromatographic methods, its versatility, rapidity, and low cost make it a popular method. The traditional TLC method is gradually being replaced by HPTLC, which uses new stationary phases and automatic densitometric detection. HPTLC is a method competitive with HPLC in the analysis of drug impurities [19, 20]. 

TLC is not the best chromatographic technique for quantitative analysis and, although it can provide quantitative results, the necessary procedure tends to be more cumbersome and tedious compared with other chromatographic methods. Furthermore, for accurate work, expensive scanning equipment is required, which, as already discussed, rather reduces the cost advantage of the technique. 

For self-labelling with T, high partial pressures of tritium gas and gaseous or finely dispersed organic compounds are favourable. The products can be separated by gas chromatography or other chromatographic methods. Specific activities of the order of about 10 Bq/g are obtained. 

The correct choice of a solvent system is of paramount importance for a successful CPC separation. Selection of a two-phase (biphasic) solvent system for CPC is similar to choosing the solvents for other chromatographic methods such as for a normal column or HPLC (50). Important criteria are the polarity of the sample components and their solubility, charge state, and their ability to form complexes, etc. The most critical points in selecting solvent systems for CPC are two-fold one is solubility of the sample and the second is the difference in partition coefficients of the molecular species that are to be separated. 

Sample preparation strategies therefore tend to be very application orientated. When developing methods, it is advantageous to work in a logical and stepwise manner but recognising that potential problems are interlinked. As with other chromatographic methods, the use of internal standards closely related chemically to the analyte(s) of interest are extremely... 

A look at Table 3.2.2 shows that quantitative paper and thin-layer chromatography, above all in the microgram-range, are comparable with other chromatographic methods or techniques used in inorganic trace analysis (Table 1.3). 

One of the most important applications of frontal chromatography is the determination of equilibrium adsorption isotherms. It was introduced for this purpose by Shay and Szekely and by James and Phillips [4,5], The simplicity as well as the accuracy and precision of this method are reasons why the method is so popular today and why it is often preferred over other chromatographic methods e.g., elution by characteristic points (ECP) or frontal analysis by characteristic points (FACP) [6,7]. Frontal chromatography as a tool... 

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