Salt mobilization

Table 2 Performance Characteristics of the Spray Impact Detector for Inorganic Salts Mobile Phase, Boiled Distilled Water...

Fig. 2 CCC separation of water-soluble vitamins by cross-axis CPC. Experimental conditions sample, riboflavin sodium phosphate (2.5 mg)-I-cyanocobalamin (2.5 mg)-i-pyridoxine hydrochloride (2.5 mg)-i-thiamine nitrate (2.5 mg) solvent system, 1-butanol and aqueous 0.15 M monobasic potassium phosphate containing 1.5% of 1-octanesulfonic acid sodium salt mobile phase, lower phase flow rate, 0.2 mL/min. For other experimental conditions, see Fig. 1 caption. SF = solvent front.

A technique called salt mobilization may be used to elute the focused protein bands. The field is turned off, and one of the reservoir solutions is changed to a solution containing acid (or base) plus a different cation (or anion) than H+ (or OH ). When the electric field is turned on again, electroneutrality causes a deficiency of either H+ or OH to enter the capillary. The result is that the entire pH gradient moves toward the cathode if OH- is deficient, or toward the anode if H+ is deficient, and the focused pattern is eluted through a detector. 

Figure 12.13 shows such a separation, using a 40 cm, 75 pm uncoated fused silica capillary operated at 30 kV. A solution of ampholytes (5%) and sample (1 mg/mL of each protein) in the running buffer are loaded throughout the capillary. The application of the electric field results in slow, continuous separation and detection due to the slow electroosmotic flow. With this method, no salt mobilization is required. 

Capillary isoelectric focusing was performed in a 75-pm i. d. capillary where the surface silanols had been derivatized with linear polyacrylamide. A mixture of proteins having pi values between 6 and 8 were focused using ampholytes covering the pH 3-10 range, using an anolyte of 20 mAf HC1 and a catholyte of 20 mM NaOH. A static separation pattern was indicated by a current minimum that occurred after 90 min at 15 kV. How can salt mobilization be used to elute the separation pattern at the anodic end of the capillary ... 

HP-SEC, also called high-performance gel-permeation chromatography (HP-GPC), is performed on porous stationary phases and separates analytes according to their molecular mass or their hydrodynamic volume. As a nonretentive separation mode, HP-SEC is usually operated with isocratic elution using aqueous low salt mobile... 

The HPLC of large biomolecules such as proteins and DNA often requires specialized columns packed with wide-pore polymer or silica-based bonded phase with extra-low silanol activity.1215 Alternate approaches are pellicular materials or very small nonporous particles. Some of these columns are packed in PEEK or titanium hardware to allow the use of high-salt mobile phase and to prevent possible protein denaturing by metallic leachates. Further details on bio-separations and application examples are discussed in Chapter 7. 

Chromatography of proteins often requires the use of high-salt mobile phase, which is corrosive to stainless steel.7 HPLC systems and columns for this application often use titanium or PEEK to substitute for stainless steel components in their fluidics. Other systems have built-in column switching for isolating active components using affinity/IEC or other tandem two-dimensional chromatography (Figure 4.13d). 

Since general purpose CE instruments are only equipped with a single point on-column detector, which is usually located close to one end of the capillary column as shown in Figure 19.1c, these stationary protein zones within the column have to be mobilized past the detection point of the single point detector. For cIEF performed on these CE instruments (conventional cIEF), a mobilization process is necessary following the focusing process [10,11]. There are three ways of performing the mobilization pressure-driven mobilization [11], salt mobilization [3], and EOF-driven mobilization [12]. These three methods can be used in any combination. 

With salt mobilization, at the end of the focusing process salts are added into one of the electrolytes. The addition of the salts creates a pH shift at this end of the column under the separation voltage, and then the shift gradually progresses deeper into the column. This causes the whole pH gradient within the column to shift toward this end of the column. 

A CO2/N2 selectivity of around 1,800 for cross-linked polyvinyl alcohol polymeric membranes containing amine functionality in the form of 2-aminoisobutyric acid-potassium salt (mobile amine carrier) and poly (allylamine) (fixed carrier), at 110 °C was reported by Zou et al. [13], The main differences between this study and that reported by Zou et al. [13] are as follows (1) they used two types of amine carriers while suggesting that the mobile carriers connibuted more to the CO2 flux than the fixed carriers as opposed to APTS-modified membranes where the carriers were fixed [attached to the pore walls], (2) in their study, water was used both on the feed and permeate side, enhancing the reaction rate of CO2 with... 

Research by Bai et al. [12] reported CO2/N2 selectivity of 249 at the same experimental conditions and feed gas composition as Zou et al. [13], namely 100 °C and feed pressure of 30 psi for polymeric membranes using sulfonated polybenzimidazole (SPBl)-ethylenediamine (EDA) copolymer containing 30 % SPBI, 50 % polyethylenimine (PEI) (hxed amine carrier), and 20 % 2-aminoisobu-tyric acid-potassium salt (mobile amine carrier). In this case again, they had mobile and fixed amine carriers. 

II. 4. Micellar Bile Salt Mobile Phases a) Bile Salt Description and Properties... 

Figure 13.5 shows that the steroid skeleton of the molecule has a polar side and a lipophilic apolar side. Micelle formation is possible with a significant solubilization power for apolar molecules. The bile salt cmc depends on the pH and ionic strength. It is approximately 5-10 mM for cholic acid and 3-5 mM for the deoxycholic acid [11]. The great and appealing property of the bile salt molecules is their chiral structure with 3 or 4 asymmetric centers. Investigating micellar bile salt mobile phase in LC, it was hoped that their micelles would be able to distinguish enantiomers. 

Hinze was the first to investigate the capabilities of micellar bile salt mobile phases [11, 12]. He found that a significant amount ( 5% v/v) of a long chain n-alcohol (pentanol, hexanol or heptanol) was useful to minimize the bile salt adsorption on the C18 stationary plmse. A wide range of solutes could be separated by these phases, PAHs, quinones, steroids, indoles, polar and lipophilic vitamins. These phases were also able to resolve optically big enantiomers such as binaphthyl derivatives [12]. Such compounds are... 

R.W. Williams, Jr., Z.S. Fu and W.L. Hinze, Micellar Bile Salt Mobile Phases for the LC Separation of Routine Compounds and Optical, Geometrical and Structural Isomers, J. Chromatogr. Sci., 28 292 (1990). 

W. Hu, T. Takeuchi and H. Haraguchi, Retention Mechanism of Enantiomeric Separation by LC with Micellar Bile-Salt Mobile Phases, Chromatographia, 33 63 (1990). 

Today, lEC is used infrequently in comparison with other chromatographic methods. In most cases, IPC is more convenient because of its higher column efficiency, more stable and reproducible columns, and easier control over selectivity and resolution. There are, however, cases for using lEC instead of RP- or IP-HPLC, especially when organic ions have poor UV absorbance and need other detection (conductivity or MS). Then, completely volatile components of mobile phase are required. In such cases, lEC with volatile buffers fulfil this requirement, whereas ion-pair reagents are not sufficiently volatile in most cases also, when compounds are isolated or purified by HPLC separation, the removal of mobile phase is necessary. When multistep separation is required, the aqueous buffer-salt mobile phase used for ion-exchange allows direct injection of a sample fraction onto an RP column for the next step of separation. This may be difficult with IP systems. 

In an attempt to compensate for the acidosis, bone salts are mobilized. Unless the patient is treated with alkalies, bone salt mobilization results in osteomalacia. Calcium is excreted in the urine, and the combination of high calcium levels, high pH, and low citrate concentration in the tubular fluid facilitates precipitation of calcium salts, thus leading to nephrocalcinosis. Secondary hyperparathyroidism develops as a consequence of the negative calcium balance. The inability to concentrate urine and the accompanying polyuria result in part from the hypokalemia and in part from the hypercalciuria. 

Figure 8 Thin-layer chromatograms of the mixture of DAM salts, mobile phase acetome-chloroform (3 1), Reagents (a) DAM, (b) MDAM (c) HDAM (d) PDAM Anions 1,2,3,4,5,6, and 7 refer to Sol , Cl", Br, N(, SCN", r and CIOJ respectively 8 reagent (Reproduced from Ref. 143 by permission of Friedr. Vieweg and Sons Verlagsgesellschaft mbH).

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