Mobile phases volume

As the (n)th plate of the column acts as the detecting cell, there can be no heat exchanger between the (n-l)th plate and the (n)th plate of the column. As a consequence, there will be a further convective term in the differential equation that must account for the heat brought into the (n)th plate from the (n-l)th plate by the flow of mobile phase (dv). Thus, heat convected from the (n-l)th plate to plate (n) by mobile phase volume (dv) will be... 

Thus, for a packed column length (L), radius (r), with a mobile phase volume equivalent to 60% of the column volume,... 

To save costs, only the mobile phase volume intended for use should be calculated and prepared. Typically, the lower end of the plate should be immersed several millimeters, i.e., just about 4 mm, for preparative purposes. 

In NPLC, which refers to the use of adsorption, i.e. liquid-solid chromatography (LSC), the surface of microparticulate silica (or other adsorbent) constitutes the most commonly used polar stationary phase normal bonded-phase chromatography (N-BPC) is typified by nitrile- or amino-bonded stationary phases. Silica columns with a broad range of properties are commercially available (with standard particle sizes of 3, 5 and 10 im, and pore sizes of about 6-15nm). A typical HPLC column is packed with a stationary phase of a pore size of 10 nm and contains a surface area of between 100 and 150m2 mL-1 of mobile phase volume. 

In this equation, the constant Kz, which can be easily inferred from the intercept, represents a system-specific constant that is related to the ion-exchange equilibrium constant K(Lmol ), the surface area 5 (in m the charge density on the surface, that is, the number of ion-exchange sites qx available for adsorption (in molm ), and the mobile phase volume Vo (in L) in the column as described by the following equation ... 

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. 

However, by no means are microbore HPLC columns nonoptimal when sample volumes are limited. Because of the substantially reduced mobile-phase volumes necessary to carry out a given separation, microbore columns are more easily interfaced to many useful detection systems, for example, electron-capture detectors, nitrogen-specific thermionic detectors, and mass spectrometers. 

Kp may be related to the retention volume, the mobile phase volume, and the stationary phase volume. The total moles of substance injected onto the column, n, is divided between the stationary phase and the mobile phase,... 

Let us now examine the chromatogram produced by this separation. Starting at the point of injection, we follow the baseline to the first deflection. After about 2min, we see a small positive deflection immediately followed by a small negative deflection, which then returns to the baseline. The center of this peak complex is called the void volume (Vo). It represents the amount of mobile phase contained inside the column, but outside the packing material. It is the mobile phase volume necessary to wash out the sample solvent. 

The next peak is that produced by the blue dye (B) we will measure the mobile phase volume at the center of the peak and call it VB. In the same manner, we can calculate VA as the retention volume for the red dye. We could measure the distances Vo, VA, VB just as easily in minutes since injection or as centimeters of graph paper. As we will see, the separation parameters are... 

Retention Time—The time or mobile phase volume need to elute and detect a component of the mixture in a detector. 

If open columns are used, then the phase ratio will vary with the column diameter (provided that the film thickness is kept constant). The cross-sectional area of the column (and hence the mobile phase volume) is proportional to the square of the column diameter, while the wall area is proportional to the diameter itself. Hence, the phase ratio is inversely proportional to the column diameter. 

The effect of limited penetration of the pores by the largest molecules may also be applied beneficially for the separation of very large molecules. Depending on the size of the molecules (in solution), they will be more ore less excluded from the pores, and hence the retention times will be affected. This effect is used in size exclusion chromatography (SEC) or gel permeation chromatography (GPC). In this technique, any interactions between the solute molecules and the stationary phase are purposefully avoided. The solute molecules remain exclusively in the mobile phase, but the accessible mobile phase volume, and hence the retention volume, may vary between the total volume of the mobile phase and the so-called exclusion volume, which is the total volume of mobile phase outside the pores. The latter elution volume applies to very large solute molecules (excluded solutes),... 

The retention volume (or time) that has the mobile phase volume subtracted out is of interest for theoretical work. 

For the assessment of the extent of change of the phase ratio of a HPLC column system with temperature or another experimental condition, several different experimental approaches can be employed. Classical volumetric or gravimetric methods have proved to be unsuitable for the measurement of the values of the stationary phase volume Vs or mobile phase volume Vm, and thus the phase ratio ( = Vs/Vm). The tracer pulse method266,267 with isotopically labeled solutes as probes represents a convenient experimental procedure to determine Vs and V0, where V0 is the thermodynamic dead volume of the column packed with a defined chromatographic sorbent. The value of Vm can be the calculated in the usual manner from the expression Vm = Eo — Vs. In addition, the true value of Vm can be independently measured using an analyte that is not adsorbed to the sorbent and resides exclusively in the mobile phase. As a further independent measure, the extent of change of 4> with T can be assessed with weakly interacting neutral or... 

Column volume. A volume of solvent equal to the volume of the column occupied by the mobile phase. A column volume is also referred to as the void volume of the column or the mobile phase volume. 

Mobile phase volume. The volume of solvent in a packed column, given by the amount of mobile phase required to elute a sample component which does not interact with the packing material (VM also known as the void volume, T0)-... 

Void volume. The total mobile phase volume in a packed column. The volume between the packing particles (interstitial volume) and the volume within the packing pores added together equal the void volume, V0- Void volumes are typically 40-80% of the empty column volume and are determined by injecting a nonretained component, for example, heptane on a silica column, using chloroform as the mobile phase. The void volume is also referred to as the mobile phase volume, VM-... 

According to equation (8-3), the retention volume for a solute is equal to (1 + k ) multiples of the columns mobile phase volume VM The k value is a unitless relative retention measurement which can be calculated from experimental values. Rearrangement of equation (8-3) yields... 

In gas chromatography the analyte partitioning between mobile gas phase and stationary liquid phase is a real retention mechanism also, phase parameters, such as volume, thickness, internal diameter, and so on, are well known and easily determined. In liquid chromatography, however, the correct definition of the mobile-phase volume has been a subject of continuous debate in the last 30 years [13-16]. The assumption that the retardation factor, i /, which is a quantitative ratio, could be considered as the fraction of time that components spend in the mobile phase is not obvious either. 

Figure 2-5. Illustration of the column shce for construction of mass balance. Mobile-phase flow F in mL/min analyte concentration c in mol/L n is the analyte accumulation in the shce dx in mol v is the mobile-phase volume in the shce dx expressed as VqIL, where L is the column length s is the adsorbent surface area in the shce dx, expressed as S/L, where S is the total adsorbent area in the column.

The concept of the void volume and mobile-phase volume was briefly discussed in Chapter 1, Section 1.7.2. In the partitioning model (Section 2.8) the total volume of the liquid phase in the column is equal to the sum of the volumes of the mobile and stationary phase in the same column. 

The analyte molecules are distributed between the mobile phase, the acetonitrile adsorbed layer, and the adsorbent surface. The analyte could be in neutral, ionic, and ion-associated form, assuming that only neutral and ion-paired analyte could partition into the organic adsorbed layer and subsequently be adsorbed on the surface. This discussion is limited to the hypothetical energetically homogeneous surface of the reversed-phase adsorbent where residual silanols are effectively shielded by the alkyl bonded layer with high bonding density. The effect of accessible residual silanols, although much discussed in the literature, has never been estimated quantitatively in direct experiments and thus could not be included in any theoretical considerations. The total amount of analyte in the bulk solution p) is represented as a sum of the concentrations of each form of the analyte multiplied by the mobile-phase volume ... 

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