Column void volume measurement

The retention time of the non-adsorbing methane (ti) is the measure of the column void volume or holdup. Ethylene is adsorbed by the catalyst, hence it does not reach the detector until the available surface is saturated, at which point ethylene breaks through and is detected by the sensor (t2). The adsorbed volume of ethylene is given simply by ... 

Improved separation of natural oil TGs using short columns packed with 3-//m alkyl bonded-phase particles was reported by Dong and DiCesare (88). The HPLC columns used were HS-3 high-speed columns packed with 3-/um C18 bonded-phase particle (100 X 4.6-mm ID) with a column void volume of ca. 0.8 ml and efficiencies in the range of 13,000-15,000 theoretical plates (measured under optimized conditions) and HS5 C,8 columns (125 X 4.6-mm ID packed with 5-yttm particles). Two detectors were used a modified refractive index detector having an 8-/rl flow cell and 0.007-in. ID inlet tubing and a variable-wavelength UV/visible detector. 

Experimental assessment of the column void volume proved to be critical since the solute retention volume approaches the void volume as pressure is increased. Following the recommendations of Kobayashi (24), we used an unretained solute, methane, for this measurement. Values for the void volume determined over an extended pressure range were 1.8 and 0.5 ml. for the crosslinked resin and alumina columns, respectively. These figures were in excellent agreement with void volume approximations of 1.4 and 0.45 ml. based upon the geometric volume of the column assuming a porosity of 0.6 for the packed beds. 

Capacity factor, k. A measure of the interaction of a sample component with a specific packing material and solvent combination. The k term is a measure of the solvent volume required to elute a component from the column, expressed as multiples of the column void volume. 

Once the appropriate detector is chosen and the hardware system is operating correctly, the next ingredient for a successful chromatogram is retention of the components in the mixture. The measure of the retention of a compound on a column is referred to as the capacity factor and is a measure of retardation of the compound in terms of the number of column void volumes it takes to elute the apex (center) of the peak. This measure is called k (pronounced kay prime) and is simply the ratio of the elution volume of the component (V/) to the void volume of the column (Vo), which is expressed as... 

The column void volume, Vo, is dehned as the total volume of the liquid phase in the column and could be measured independently [18]. Total adsorbent surface area in the column, S, is determined as the product of the adsorbent mass and specific surface area. 

The use of the true volume of the liquid phase in the column as the void volume can lead to the principal difficulties in the interpretation of the retention of polar analytes that are also excluded from the contact with the adsorbent surface. The retention volume of these analytes will be lower than the column void volume, and thus their retention factors will be negative. A logarithm of negative retention factors does not exist that shows the applicability limit of the approximate theory described above. In a general sense the void volume should not change as a function of the type and organic composition. Table 2-1 demonstrates the compatibility of the void volume measured using different thermodynamically consistent methods. 

The void volume is the volume of the liquid phase inside the column. The importance of this parameter has been discussed in Chapter 2. Despite the very long debates, this is still a subject of significant controversy. Essentially, anyone who intends to measure the column void volume has to answer the question if he/she wants correct or estimated (convenient) measurements. 

Measurement of Void Volume. It is generally believed that the injection of so-called unretained component can be used for the measurement of the column void volume. The biggest challenge is to find a compound that is really unretained. In the last 40 years, many different analytes have been... 

These difficulties are inspiring the search for other alternative methods for the void volume measurements. The main question anyone should answer is, What should the required accuracy be For any prediction or thermodynamic-type work, the void volume values should be very accurate. For pure analytical purposes or for the comparison of different columns, the use of some markers is justifiable insofar as all experimental conditions are kept the same all the time. 

Haddad et al. measured retention volumes for a variety of bases on a quaternary ammonium functionalized PS-DVB stationary phase using dilute aqueous sodium hydroxide as the eluent [11]. Values for the retention volumes and distribution coefficients of selected bases are given in Table 8.3. Strong bases, which are fully ionized a the eluent pH, elute at the column void volume and have a value of 1.0. Solutes intermediate between these two extremes are partly ionized and generally can be separated by an ion-exclusion mechanism. 

There have been a number of publications on determination of column void-volume (30-35) and this subject is still one of the hot topics in LC. From our knowledge and basic study on this subject(36, 37), we decided sodium nitrite and sodium nitrate are good solutes to measure column void-time in reversed-phase LC. Therefoi, the elution time of sodium nitrite was used as t in this work. Its concentration used was c.a. 100 ppm in each mobile phase. 

The protocol is strai itforv d. After complete column equilibration with the surfactant solution at concentration C, the column is flushed by two or three void volumes of aqueous phase without surfactant. IThs efiftuent is not collected. This step washes out the water soluble surfactant contained in the void volume without desorbing the surfactant of the stationary phase. Next, the column is rinsed with at least 10 column void volumes of pure methanol (or propanol). The effluent, containing the desorbed surfactant, is collected. Methanol is evaporated and the dry surfactant is re-dissolved in a measured volume of water. The amount of surfactant is determined by titration. 

The difference in retention times between the 920,000 PEO and the 21,000 PEO in Table 17.9 can be used as a measure of the void or pore volume that effectively provides the linear separation range for these columns in water and in a water/methanol mixture. The better separation efficiency of the Shodex columns over the TSK columns is partially related to the larger void volumes of the Shodex columns than the TSK columns. The difference in void volumes for the Shodex, TSK GM-PW, and TSK GM-PWxl columns is partially attributed to the difference in the inner diameters of the three columns, which are 8 (Shodex), 7.8 (TSK GM-PW) and 7.5 (TSK GM-PWxi.) mm. Table 17.9 also... 

Hydrodynamic volume refers to the combined physical properties of size and shape. Molecules of larger volume have a limited ability to enter the pores and elute the fastest. A molecule larger than the stationary phase pore volume elutes first and defines the column s void volume (Vo). In contrast, intermediate and smaller volume molecules may enter the pores and therefore elute later. As a measure of hydrodynamic volume (size and shape), SE-HPLC provides an approximation of a molecule s apparent molecular weight. For further descriptions of theoretical models and mathematical equations relating to SE-HPLC, the reader is referred to Refs. 2-5. 

To assay the amount of LBP, first an excess amount of luciferin is added to the sample at pH 8 to saturate the binding site of LBP, and then the excess luciferin is removed by gel filtration using a small column of Sephadex G-25 (about 1 ml volume) also at pH 8. The luciferin-bound LBP is eluted at the void volume. To measure the amount of LBP, the following assay buffer is added to a small portion of the elu-ate 0.2 M phosphate, pH 6.3, containing 0.25 mM EDTA, 0.1 mg/ml of BSA, and luciferase (Morse and Mittag, 2000). The total light obtained represents a relative amount of LBP the absolute amount (the weight or the number of molecules) cannot be obtained because the quantum yield of the luminescence reaction is not known. 

Microbial over-growth was controlled with carbon dioxide passed through the bed. There was a maximum 30% increase in the beads diameter at the lower part of column, where the glucose concentration was maximum. The void volume was measured by passing sterilised water. In addition to the carbon source, the feeding media consisted of 1 g l 1 yeast extract pumped from the bottom of the reactor, while the flow rate was constant for a minimum duration of 24 hours. 

Virial coefficients (GC) 5 Viscosity detector (SEC) 452 Viscous fingering (SEC) 447 Visualization techniques (TLC) (see derivatization (TLC)] Void volume, column (LC) 371 measurement 372 Volman trap (GC) 211 Volume of a theoretical plate 49... 

The retention factor, k, is the basic value in chromatography, and is related to the void volume (dead volume). The void volume is the space inside the column, where no retention of solutes has occurred and can be measured on a chromatogram, as shown in Figure 1.3. The void volume is about half the total volume of the column when it is packed with porous stationary phase materials. In practice, the effective void experienced by the analyte is smaller because the molecular mass of the analyte is usually much greater than that of the eluent molecule. In a model of porous stationary phase material, the pores can be represented as V-shape valleys (Figure 3.8), where region a is a support, such as... 

One unique challenge in using SCILs as stationary phases in HPLC is the determination of the column-void or mobile-phase volume. Accurate determination of retention factors, k, requires measurement of fg, the void volume. 

In a column packed with a swollen gel, two solvent phases may be distinguished, one within the gel (the stationary phase), and the other outside (the mobile phase). The volume of solvent in the gel is known as the internal volume, designated V4, and the volume of solvent outside the gel particles is the void volume of the column, designated V0. A solute will distribute itself between the two phases to an extent measured by the distribution coefficient Kd, a constant determined by the nature of the solute, the solvent, and the gel, but independent of column geometry.27 The volume of solvent required for eluting the solute from the column in maximum concentration is called the elution volume and is designated Ve it is equal to the sum of the void volume and the volume of the stationary phase available to the solute, given by Kd V. Hence,... 

The injected volume corresponding to one bolus is usually quite small, equivalent at the best to the void volume of the column. The principal advantage of the repeated bolus method is that small injection volumes are required. On the other hand, perfused activity measurements using small volumes requires high activity generators. 

Void volume is measured by passing a large, inert molecule through the column." Its elution volume is defined as F0. Blue Dextran 2000, a blue dye of molecular mass 2 X 106, is commonly used for this purpose. The volume Vm can be calculated from the measured column bed volume per gram of dry gel. For example, 1 g of dry Sephadex G-100 produces 15 to 20 mL of bed volume when swollen with aqueous solution. The solid phase occupies only 1 mL of the bed volume, so Vm is 14 to 19 mL, or 93-95% of the total column volume. Different solid phases produce widely varying column bed volumes when swollen with solvent. 

Void Volume This is the total space surrounding the gel particles in a packed column. This value is determined by measuring the volume of solvent required to elute a solute that is completely excluded from the gel matrix. Most columns can be calibrated for void volume with a dye, blue dextran, which has an average molecular mass of2,000,000 daltons. 

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