Column pore size

TSK-PW column Pore size (A) Molecular weight exclusion limit... 

GPC techniques are applicable to a wide variety of solute materials, both low and high molecular weight, dissolved in solvents of varying polarity. The selection of column type, column pore size, solvent, and temperature must be appropriately made for each solute. Care must be taken to avoid reaction between the solute and the columns or other adsorption phenomena, especially when two solvents are used—one to dissolve the solute and one in the chromatograph. Changing from one solvent to another in the chromatograph can take 24 hr before the baseline stabilizes. 

Sample analysis was performed by using an Applied Biosystems (Foster City, CA) API 3000 triple quadrupole mass spectrometer equipped with a TurboIonSpray source and an Agilent 1100 capillary HPLC system (Palo Alto, CA). The capillary HPLC system included a binary capillary pump with an active micro flow rate control system, an online degasser, and a microplate autosampler. The analytical column was a 300 pm I.D.x 150 mm Zorbax C18 Stablebond capillary column (pore size 100 A and particle size 3.5 pm). The injection volume was 5 pL, and a needle ejection rate of 40 pL/min was used. The pLC flow rate was 6 pL/min. In order to minimize dead volume before the column, the autosampler was programmed to bypass the 8 pL sample loop 1.5 min after injection. The mobile phase consisted of (A) 2 mM ammonium acetate (adjusted to pH 3.2 with formic acid) in 10 90 acetonitrile-water, and (B) 2 mM ammonium acetate in 90 10 acetonitrile-water. The percentage of mobile phase B was held at 32 % for the first minute, increased to 80 % over 8 min, and then increased tol00% over the following 1 min. 

General. Commercially available polyimides were used (see Table I). Solvents were reagent grade or better and were used as received. 1-Methylpiperazine (Aldrich Chemical Co.) was > 99.9% pure. Lactic acid (Aldrich) was reduced to 50% by weight in water and refluxed for an hour to hydrolyze esters. IR spectra were recorded on a Perkin-Elmer Model 1430 spectrometer. Thermogravimetric analysis was done on a P-E System 4/TGS-2 instrument. Size exclusion chromatography was done on a Perkin-Elmer Series 3B equipped with the LC-75 spectrophotometric detector. The column set used consisted of P-E 0258-2134, 2133, and 2131 columns (pore sizes 103, 104, and 106 A, respectively). For electrophoretic deposition experiments, a TCR Power Supply (Electronic Measurement Systems Inc.) was used. Temperatures are reported in °C throughout. 

The liquid chromatogram of a polydimethylsiloxane (PDMS) sample with OH end groups is shown in Figure 31.3a. The separation was achieved by using a Nucleosil silica column (pore size 120 A, 200 x 2.1 mm ID). The mobile phase was n-hexane/ethyl acetate, the flow rate 0.4 mL/min. ... 

The critical solvent composition (esc) for PDMS standards was determined using a silica column (pore size 100 A, average particle size 5 pm, 200 x 4 mm ID) and toluene/Ao-octane mixtures. When plotting the retention factor k versus solvent/nonsolvent composition, the esc is indicated by the intersection point demonstrated in Figure 31.4a. Under these conditions, PDMS samples of different molecular weights elute at the same retention time. Any separation of an unknown sample into different peaks can be ascribed to a heterogeneity other than molecular weight distribution. 

A number of fundamental questions related to chromatographic equilibria and dynamics have been explicated. The fundamental issue of the nature of the macromolecular dimensions that determine Ksfc s the subject of Chapter 1. The resolving power of aqueous SEC columns is considered from the perspective of column pore size distributions in Chapter 6. These and other aspects of efficiency are explored in detail in Chapter 7. Interactions between macromolecules and stationary phases are covered in both Chapter 2 (hydrophobic effects) and Chapter 3 (electrostatic effects). 

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. 

Fig. 8 Change in the elution time of dextrin with various molecular weights in response to temperature changes from the PNIPAAm-treated column (pore size 23.7 nm, flow rate 0.6 ml/min).

The significance of the various columns is explained in the notes below the table, which enable the calculations of 6v l6r to be followed through. Only the first few lines are reproduced, by way of illustration the pore size distribution curve resulting from the complete table is given in Fig. 3.18 (Curve A), as a plot of 6i j6r against f. 

Two classes of micron-sized stationary phases have been encountered in this section silica particles and cross-linked polymer resin beads. Both materials are porous, with pore sizes ranging from approximately 50 to 4000 A for silica particles and from 50 to 1,000,000 A for divinylbenzene cross-linked polystyrene resins. In size-exclusion chromatography, also called molecular-exclusion or gel-permeation chromatography, separation is based on the solute s ability to enter into the pores of the column packing. Smaller solutes spend proportionally more time within the pores and, consequently, take longer to elute from the column. 

Separation of C oand C70 can be achieved by HPLC on a dinitroanilinopropyl (DNAP) silica (5pm pore size, 3(X)A pore diameter) column with a gradient from H-hexane to 50% CH2CI2 using a diode array detector at wavelengths 330nm (for C q) and 384nm (for C70). [J Am Chem Soc 113, 2940, 1991.]... 

The stationary phase constitutes about 12% of the column volume, which is equivalent to only about 17% of the mobile phase content of the column. The values given in Table 2 are probably representative of most reverse phase columns but will differ significantly with extremes of pore size and pore volume. 

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.]... 

Irrespective of the development of media, many of the traditional media are successfully defending their position. This is due to their hydrophilic nature, preserving biological function of the separated molecules, but also the fact that columns may be prepared easily and, finally, some of the classical media, e.g., Sephadex, have a selectivity that is so far unsurpassed and therefore very fit for use. Intersting enough, Sephadex is still the premiere gel filtration medium for desalting due to the optimal pore size and particle size of this medium (see Section II,C). 

Based on the requirements of the separation, media of suitable pore size, particle size, and surface properties are selected as well as column dimensions and column material. In some cases a suitable combination of media type and column dimensions may be available as a prepacked column. In most cases, this is a more expensive alternative to preparing the column yourself but will provide a consistent quality as assured by the manufacturing and testing procedures of the vendor. The consistent quality may be critical in obtaining reproducible results and may thus be a cost-effective solution. Also, the fact that smaller particle-sized media are more difficult to pack and require special, and expensive, equipment has resulted in that gel filtration media of small particle size, e.g. smaller than 15 /zm, are predominantly supplied as prepacked columns. 

Generally, optimizing the selectivity by choosing a gel medium of suitable pore size and pore size distribution is the single most important parameter. Examples of the effect of pore size on the separation of a protein mixture are given in Fig. 2.15. The gain in selectivity may then be traded for speed and/ or sample load. However, if the selectivity is limited, other parameters such as eluent velocity, column length, and sample load need to be optimized to yield the separation required. 

Select the appropriate chromatographic column or columns. Choose a column packing with a pore size that will resolve the molecular size range of the sample. 

Zorbax PSM Bimodal and Trimodal columns are packed with mixed pore-size packing to achieve linear size separations over a broad molecular weight range (Table 3.3). Zorbax PSM Bimodal columns are packed with PSM 60 and PSM 1000 particles, and Trimodal columns contain PSM 60, PSM 300, and PSM 3000 particles (Fig. 3.4). Carefully selecting and mixing different pore-size particles in columns provide much better linearity than coupling columns that are each packed with single pore-size particles. 

Resolution increases when columns of the same pore size are used in series however, increasing the number of columns in series also increases the total... 

As with other size-exclusion techniques, the pore size of the selected Zorbax GF column should provide resolution over the molecular size range of the proteins that are to be separated. The Zorbax GF-250 column separates proteins in the range of 4000 to 400,000 Da. The Zorbax GF-450 provides separation over the range of 10,000 to 1,000,000 Da. When these two columns are coupled, they can be used to separate proteins with molecular weights of 4000 to 1,000,000. 

The different pore sizes and exclusion limits of each TSK-GEL SW column will have a substantial effect on the resolution of a biomolecule mixture. G2000SW packing, which has the smallest pores, provides the best resolution for smaller proteins such as myoglobin and cytochrome c (16,900 and 12,400 Da, respectively, Rs = 1.01). Resolution of the proteins myoglobin... 

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