Column equilibration

Equation (25) can be extended to provide a general equation for a column equilibrated with (q) solutes at concentrations Xi, X2, X3,...Xq. For any particular solute (S), if its normal retention volume is Vr(S) on a column containing (n) plates, then from the plate theory, the plate volume of the column for the solute (S), i.e., (vs) is given by... 

Method development remains the most challenging aspect of chiral chromatographic analysis, and the need for rapid method development is particularly acute in the pharmaceutical industry. To complicate matters, even structurally similar compounds may not be resolved under the same chromatographic conditions, or even on the same CSP. Rapid column equilibration in SFC speeds the column screening process, and automated systems accommodating multiple CSPs and modifiers now permit unattended method optimization in SFC [36]. Because more compounds are likely to be resolved with a single set of parameters in SFC than in LC, the analyst stands a greater chance of success on the first try in SFC [37]. The increased resolution obtained in SFC may also reduce the number of columns that must be evaluated to achieve the desired separation. 

The molecular weight distribution of cell wall polysaccharides was estimated by gel filtration with a TOSOH TSK gel G4000 PWXL (7.8 x 300 mm) column equilibrated and eluted with 0.05 M sodium acetate, 0.01 M EDTA, 0.05 M NaCl (pH 5.0) in polyuronide and 0.05 M sodium citrate, 0.1 M NaCl (pH 5.5) in the hemicellulose fraction. Samples (1 mg/ml) of 100 ml were injected. The eluate was monitored by a refractive index detector (Shimadzu R1D-6A, Kyoto, Japan) and collected at the fraction size of 0.4 ml. 

Gel Filtration. The lyophilized protein was redissolved in 50 mM phosphate buffer, pH 7.4 0.15 m NaCl 0.013 % sodium azide and loaded on a Superdex 75HR1030 column equilibrated with the same buffer. Elution was downward flow (0.15 ml/min) and 0.25 ml fi actions were collected. Fractions with pectin lyase activity were combined, dialyzed against distilled water and used in the next step. To estimate the molecular mass of PNL, the column was calibrated with standard proteins (Sigma MW-GF-70 Albumin, 66,000 Da Carbonic Anhidrase, 29,00 Cytochrome, 12,400 and Aprotinin, 6,500). The proteins were eluted in the conditions described above and their volumes (F ) were calculated fi om the peak maximum of the absorbance at 280 nm. The partition coefficient was obtained fi om the relationship where F, represents the bed volmne of column and F the void volume (which was calculated using blue dextran. Sigma). The molecular mass was determined using a standard curve of vs the logarithm of the molecular masses of the standards [28, 29]... 

Figure 2. Gel filtration. The dry residue obtained after ammonium sulfate precipitation was redissolved in 50 mM phosphate buffer, pH 7.4 0.15 M NaCl 0.013 % sodium azide, which was loaded on a Superdex 75HR1030 column equilibrated with the same buffer. Elution was downward flow (0.15 ml/min) and 0.25 ml fractions were collected. The fractions were assayed for protein content (— ) and PNL activity (- - ).

Hydroxy-apatite chromatography was carried out on a 1.7 ml HA-Ultrogel (IBF) column equilibrated in 10 mM phosphate buffer (pH 6). Elution was performed with a linear 10-300 mM phosphate buffer (pH 6) gradient. 

Cleanup procedures for acetamiprid, IM-1-2 and IM-1-4. Dilute the concentrate with 10 mL of distilled water and apply the solution to an Extrelut 20 column, equilibrate for 20 min at ambient temperature and pass 100 mL of dichloromethane through the column. Collect the eluate and add 0.5 mL of diethylene glycol and then concentrate the dichloromethane to about 0.5 mL by rotary evaporation. Prepare the HPLC-ready sample solution by dissolving the residue in 50% aqueous acetonitrile. 

Experiment 1 The load and column equilibration buffer were adjusted to 2 M ammonium acetate. The DMT-on product was eluted in 0.5 M ammonium acetate followed by a linear gradient of 0 to 0.5 M ammonium acetate. 

The sample is loaded at a flow-rate of 1 ml/min onto the FPLC column equilibrated with the same MOPS buffer used to resuspend the RNA pellets. The free nucleotides are completely removed with a 5-ml wash with 350 mM NaCl and the RNA is eluted with a 20-ml (350—750 mM NaCl) linear gradient and analyzed by PAGE/urea gel electrophoresis (see later). Up to 2 mg of RNA can be loaded onto and eluted from a 1-ml (of resin) mono Q column without loss of resolution. The homogeneity of RNA in the fractions collected, as seen by gel electrophoresis, should be >90%. The appropriate fractions are pooled and the RNA collected by ethanol precipitation. The RNA pellet is washed twice with 70% ethanol, air-dried, and finally redissolved in DEPC-treated H20. The total recovery after the entire procedure of purification is = 90%. This protocol yields = 800 pmoles of purified 002 mRNA/pmole template DNA. 

Analytical size exclusion chromatography was performed on an Akta Purifier (Pharmacia) using a 24-ml Superose 6 HR 10/30 column (Pharmacia). 250-pl samples in AP-buffer (20mM Tris buffer, pH 8.0, 100 mM NaCl), with or without 0.1 % P-DM, were injected onto the column equilibrated with the same buffer and eluted at a flow rate of 0.3 ml min 1. Elution profiles were recorded at 220, 280 and 674 nm. 

For comparison of similar columns, it is important that experimental conditions for the test chromatogram are faithfully reproduced and sufficient time is allowed for column equilibration before starting the test. The expected changes in column performance parameters that are due to changes in the experimental conditions are summarized in Table 15.13. 

Briefly, liposomes (10mM) were incubated for 30minutes at 37°C for egg phosphatidylcholine (EPC) and at 60°C for HSPC-based liposomes with 50 X 10 dpm of methylamine (1 x 10 dpm/mole). At the end of incubation an aliquot of this mixture was passed down a Sephadex G-50 minispin column equilibrated in 10 mM histidine-sucrose buffer 10%, pH 6.7 buffer. Liposomes were eluted at the column void volume and separated from the unencapsulated methylamine. The concentration of liposomes in the original liposomal dispersion and in the void volume fraction was determined from the organic phosphorus (phospholipid) concentration (see section Lipid Quantification and Chemical Stability above) (10,49,53). 

Adsorption-desorption coefficients are determined by various experimental techniques related to the status of a contaminant (solute or gas) under static or continuous conditions. Solute adsorption-desorption is determined mainly by batch or column equilibration procedures. A comprehensive description of various experimental techniques for determining the kinetics of soil chemical processes, including adsorption-desorption, may be found in the book by Sparks (1989) and in many papers (e.g., Nielsen and Biggar 1961 Bowman 1979 Boyd and King 1984 Peterson et al. 1988 Podoll et al. 1989 Abdul et al. 1990 Brusseau et al. 1990 Hermosin and Camejo 1992 Farrell and Reinhard 1994 Schrap et al. 1994 Petersen et al. 1995). 

Elevated Temperature Studies. The Varian 5000 liquid chromatograph and a Waters Associates 401 differential refractometer were employed. The column was heated with a Varian universal heater block at an estimated accuracy of 0.5 C. About 15-30 minutes were allowed for column equilibration for a given temperature. The recorder employed was a Varian 9176. 

Because of the problems encountered with the water system, the use of aliphatic alcohols, ie.g., methanol, ethanol, and isopropanol, as modifiers of the adsorption strength has been recommended (44. 45. 50. 51). Usually, between 0.01 and 0.5% (v/v) alcohol is added to the eluent. As an example, the k values for the benzyl alcohols on a silica column are in the same range when eluted with dichloromethane containing either 0.1% water (50% water-saturated) or 0.15% methanol or 0.3% isopropanol (45). The preparation and preservation of these alcohol-eluent mixtures is accompanied by problems similar to those discussed with water-modified eluents. Also, column equilibration is slow (44). The efficiency of columns operated with alcohol-modified eluents is generally lower than that of water-modulated eluent system. At some alcohol concentrations, distorted peaks with tailing or frontal asymmetry have been observed 44), but olhei workers using another silica could not verify this observa tion (61). 

Add the cell lysate to a prepacked MIcroSpIn GST or GSTrap FF column equilibrated with the binding buffer. 

Major disadvantage of gradient IPC is slow column equilibration after changing the mobile phase. Complete washout of the adsorbed ion-pair reagent from the column may be difficult to achieve. Hence, it is not advisable to use a column in RPC without ion-pair reagents once it was run in the IPC mode. 

Equations 10.47 through 10.49 constitute the basis for the method of isotherm determination by the perturbation method. The retention time of a small injection of a solute in a column equilibrated... 

Dissolve 4 mg of the carrier protein in 400 pi Soln. A. Dissolve about 0.5 mg SMCC (succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carhoxylate, Mr 334.3) in 50 pi DMF and add this solution to the carrier protein. Shake at RT for 1 h, centrifuge and desalt on a Sephadex G-25 column, equilibrated with Soln. B (the activated carrier appears in the void volume). Cool the receiving tube in an ice bath. 

Dialyze an antibody solution against Soln. C and concentrate to 20-30 mg/ml. Add 3 mg of sulfo-SMCC (3-sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-l-caboxylate, Mr 436.7) or of sulfo-GMBS (N-(y-maleimidobutyryloxy)-3-sulfo-N-hydroxysuc-cinimide ester, Mr 382.3), dissolved in ddH20 to 60 mg/ml, to 10 mg of antibody, rock at RT for 15 min and add further 3 mg of coupling reagent. Desalt on a Sephadex G-25 column, equilibrated with Soln. A, after total incubation time of 30 min and use the activated antibody (protein) for conjugation immediately (see above). 

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