Void volume, columns

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. 

1 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 

The simplest possible solution is to use a single-component eluent for example, inject deuterated acetonitrile in the flow of neat acetonitrile. The isotopic effect (the difference in the retention of molecules with different isotopes), although it exists, could be assumed negligible. 

All these methods require the use of the refractive index of mass spectrometry (MS) detectors, since the detection of the isotopes is not possible with conventional ultraviolet (UV) detectors. 

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. 

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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 ... 

As noted before, the whole spectrum of particle sizes between 38 and 357 nm is encompassed with a AV of U.O ml or about 6% of the total column void volume. This low capacity of the HDC system is counterbalanced by its excellent resolution both of itself and in comparison to porous packing systems. The latter point is addressed in the next section. 

A large number of experimental methods have been proposed to estimate the column void volume without any single method emerging... 

Column Void Volume V. Retention volume corresponding to the column holdup time V. - V. 

Figure 1 shows a typical chromatogram, which includes a time axis, an injection point, and an analyte peak. The time between the sample injection point and the analyte reaching a detector is called the retention time (t ). The retention time of an unretained component (often marked by the first baseline disturbance cansed by the elution of the sample solvent) is termed void time (tg)- Void time is related to the column void volume (Vq), which is an important parameter that will be elaborated later. 

The concept of column void volume (Vg) is important for several reasons. Void volume is the volume of the empty column minus the volume occupied by the solid packing materials. It is the liquid holdup volume of the column that each analyte must elute from. Note that the void volume is equal to the void time multiplied by the flow rate (T). 

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). 

Vo = elution volume of the polymer Vo = column void volume Vt = total column volume... 

The values of IF/M calculated from equation (16.8) must be corrected for the weight of adsorbate contained in the column void volume, which can be obtained by subtracting the powder volume from the volume of the empty column. 

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. 

The column packing material surface is visualized as beads containing tapered pits or pores. As the mobile phase sweeps the injection passed these pits, the dissolved compounds penetrate, if their largest diameter (Stokes radius) is small enough to fit (Fig. 4.10). If not, they wash down the column with the injection front and elute as a peak at the column void volume, which is called the exclusion volume. 

Figure 13.3. Appearance of non-size-exclusion effects on SEC-elution curves of polyelectrolytes and other charged analytes including low-molecular-weight organic acids. Kd is the distribution coefficient and Ve, V0, and Vt are elution volume of the analyte, column void volume, and total column volume, respectively.

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. 

Compensating for the column void volume, calculate the concentration (of buffer B) at which the main component is desorbed. 

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... 

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