Columns, reversed HPLC pore size

Each column type has its own place of use. Column variety is what gives HPLC its versatility. It really depends on your compound and application. Approximately 80% of all separations are done on 5-10-jUm reverse phase Ci8 silica columns. Much of this is tradition. Reverse phase columns offer high-resolution separations for a wide variety of compounds and can be run in aqueous mobile phases. Ion exchange separations require salt solutions for separations, and these are not compatible with mass spectrometers. Size separations have lower resolving power and longer run times, but may be the only way to separate proteins solutions that will irreversibly stick to reverse phase columns. Use small pore size separation columns to remove salt from effluent from other chromatography separations. Zirconium and polymeric column are newer and offer possibilities for unique separations. 

FIGURE l.l Hydrophobic interaction and reversed-phase chromatography (HIC-RPC). Two-dimensional separation of proteins and alkylbenzenes in consecutive HIC and RPC modes. Column 100 X 8 mm i.d. HIC mobile phase, gradient decreasing from 1.7 to 0 mol/liter ammonium sulfate in 0.02 mol/liter phosphate buffer solution (pH 7) in 15 min. RPC mobile phase, 0.02 mol/liter phosphate buffer solution (pH 7) acetonitrile (65 35 vol/vol) flow rate, I ml/min UV detection 254 nm. Peaks (I) cytochrome c, (2) ribonuclease A, (3) conalbumin, (4) lysozyme, (5) soybean trypsin inhibitor, (6) benzene, (7) toluene, (8) ethylbenzene, (9) propylbenzene, (10) butylbenzene, and (II) amylbenzene. [Reprinted from J. M. J. Frechet (1996). Pore-size specific modification as an approach to a separation media for single-column, two-dimensional HPLC, Am. Lab. 28, 18, p. 31. Copyright 1996 by International Scientific Communications, Inc.. Shelton, CT.]... 

Some authors have suggested the use of fluorene polymers for this kind of chromatography. Fluorinated polymers have attracted attention due to their unique adsorption properties. Polytetrafluoroethylene (PTFE) is antiadhesive, thus adsorption of hydrophobic as well as hydrophilic molecules is low. Such adsorbents possess extremely low adsorption activity and nonspecific sorption towards many compounds [109 111]. Fluorene polymers as sorbents were first suggested by Hjerten [112] in 1978 and were tested by desalting and concentration of tRN A [113]. Recently Williams et al. [114] presented a new fluorocarbon sorbent (Poly F Column, Du Pont, USA) for reversed-phase HPLC of peptides and proteins. The sorbent has 20 pm in diameter particles (pore size 30 nm, specific surface area 5 m2/g) and withstands pressure of eluent up to 135 bar. There is no limitation of pH range, however, low specific area and capacity (1.1 mg tRNA/g) and relatively low limits of working pressure do not allow the use of this sorbent for preparative chromatography. 

Figure 11.6.3 Gradient HPLC separation of isoflavone standards (see Basic Protocol 3). Peaks 1, daidzin 2, glycitin 3, genistin 4, malonyldaidzin 5, malonylglycitin 6, acetyldaidzin 7, acetylglycitin 8, malonylgenistin 9, daidzein 10, glycitein 11, acetylgenistin 12, genistein. Conditions Waters Nova-Pak C18 reversed-phase column (150 x 3.9 mm 4-pm i.d. 60 A pore size) mobile phase 1% acetic acid in water (solvent A) and acetonitrile (solvent B) flow rate 0.60 ml/min UV detector 260 nm column temperature 25°C. The dotted line represents the gradient of solvent B.

EL-4 cells (3 x 10 ) were lysed with N,N-dimethyl-N-(3-sulfopropyl)-3-[[(3a,5 P,7a, 12a)-3,7,12-trihydroxy-24-oxocholan-24-y 1]-amino]-l-propanaminium hydroxide (CHAPS). The nuclei and membranes were pelleted and the supematent lysate filtered to remove lipids. The lysate was sequentially passed over sepharose columns containing a) normal mouse serum b) Y-3 which is an anti-K monoclonal antibody. Both columns were washed with 45 coliunn volumes of progressively lower molarity salt solutions. The beads were then treated with acetic acid to release antigen-antibody complexes and the complex was denatured by boiling in 10% acetic acid. The mixture was filtered through a 3 kDa pore-size membrane and the filtrate containing MHC class I peptides subjected to reversed phase HPLC. 

Monolithic silica is the most recently introduced class of stationary phases for electrochromatography on microchips. They benefit from their very high surface area, adjustable pore size, and controllable surface chemistry. Functionalities required for the separation in reversed-phase mode are typically incorporated onto the silica monolith by including an appropriate silicon alkoxide in the precursor mixture or by silanization of the surface after the monolith has been formed. Monolithic silica-based columns are generally known to exhibit superior performance in HPLC separations of small molecules, which is atftibuted to the presence of mesopores entailing large surface areas. 

For the preparation of analytical samples the following procedure is recommended [419]. The deproteinized biological sample (serum or tissue extract) (0.5 rql) is mixed with 1.5 ml of a 0.13% solution of o-phenylenediamine (1.33 mg ml in 3 N HCl prepared daily), 5 /il of mer-captoethanol is added, and the volume is adjusted to 3 ml with water. The mixture is heated for 30 min in boiling water. In order to terminate the reaction, the tubes are cooled on ice and 0.5 g of anhydrous sodium sulphate is added. The quinoxalinols are extracted with three 3 ml portions of ethyl acetate. The pooled extracts are dried over anhydrous sodium sulphate and evaporated to dryness. The residue is dissolved in 0.2 ml of methanol and the solution is centrifuged and filtered through a 0.45 fan pore size filter. The filtrate is ready for HPLC using a reversed phase column. 

Reverse-phase HPLC is the standard analytical tool used for the control of the purity of synthetic peptides. Analysis is most commonly performed on reversed-phase HPLC columns, consisting of 3-5 pm, 100-300 A pore size C8 or C18 silica the larger pore material is generally favoured for the analysis of larger peptides (> 30 residues). Samples are eluted with a gradient formed between water and acetonitrile. An ion-pairing reagent is added to both solvents to improve resolution and selectivity. The most popular buffer systems are listed in Table 3. 

The determination of a-carotene, trans- and cis-B-carotene from salty carrots was done by reverse-phase HPLC. All trans-, a- and B-carotene used for the generation of calibration curves were purchased from Sigma Chemical (St.Louis, MO). A Waters Nova-Pak C-18 column (pore size 4 m dimensions 3.9 mm 300 mm) with mobile phase acetonitrile methanol THF ammonium acetate (in methanol) 35 56 7 2 and flow rate 2 ml/min was used in the HPLC. Twenty-five 1 extract was injected into the column. The mobile phase was more effective in separating a-carotene from trans- and cis-B-carotene from the mixture of carotenoids compared to the mobile phase of acetonitrile ethyl acetate methanol ethyl acetate (Craft, 1992). Complete separation of trans-a-carotene from B-carotene was achieved however, only partial separation of trans- and cis-B-carotene was possible. 

Conventional reversed-jAase HPLC columns, however, need not be ignored for this area of application. Histones, for example (15), have been chromatographed on MicroBondapak Cl8, while Ultrasjrfiere ODS (Beckman Altex) with a small pore size of 8—9 nm is also often used. 

Fig. 5-6. (A) Separation of 5-C-terininal amide decapeptides (mixture RPS-POOIO [spi]) using a polystyrene, PLRP-S 100 A (Polymer Laboratories), reversed phase column with a linear gradient of 1 to 30% acetonitrile in water containing 0.1% TFA at a flow rate of l.OmLmin . Peak identification (1) Ala -Gly (free amino), (2) Gly -Oly (N -acetylated), (3) Ala -Gly (N -acetylated), (4) Val -Gly (N -acetylated), (5) Val -VaF (N -acetylated). The plot of retention time for these five synthetic amino acids as a function of pore size of the polystyrene HPLC reversed phase material is shown in (B), (1) non-porous, (2) 4000 A, (3) 1000 A, (4) 300 A, (5) 100 A pore size.

Figure 5. Anlytical HPLC separation of 300 pg venom peptides (A) and 10 ml conditioned water (B) from Conus textile. Details on preparation of the substances are given in the legend to Fig. 4. Separations were performed on an analytical CIS reverse phase column (Vydac wide pore, 4.6 x 250 mm, 5 pm particle size) at a flow rate of 0.5 ml/min. Substances were loaded on the column in aqueous 0.1% tri-fluoroacetic acid (TFA) and eluted with a linear gradient of 0-60% acetonitrile in 0.1% aqueous TFA in 0-60 minutes. On-line detection and spectral analysis was performed with a Hewlett-Packard diode array detector. The spectrum of the main peak obtained from the CW (B) is not identical to those of any of the venom derived peptides (A) that are eluted at similar times from the column (not shown). Attempts to isolate the active component(s) of Conus textile CW on reverse phase cartridge columns and Amicon filters were not successful, due to loss of the biological activity.

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