Columns characteristics

The selection of column characteristics is determined by solvent resistance, the need to visually inspect the bed, the pressure rating of the system, and the dimensions [column inner diameter (i.d.) and length (L)] required from productivity considerations. Productivity considerations will vary if the requirement is based on the amount of information per unit time (analytical gel filtration) or the amount of substance per unit time (preparative gel filtration). 

IV. Shodex OHpak SB-800HQ SERIES A. Column Characteristics... 

Classical vs. High Performance LC. Most workers are familiar with classical LC, a tool that has been predominantly used for preparative scale clean-up of samples. In order to appreciate more fully HPLC, Table I compares some of the column characteristics of classical vs. HPLC. 

The measurement of effluent volume is not very reliable because of the effect of the geometry and packing characteristics of any column. It is often more useful to use a reduced parameter, such as V/V0, which is not so dependent upon column characteristics and is comparable with the calculation for RF values in thin-layer chromatography. 

Elution of all the components of a mixture normally takes place in a finite volume controlled by the column characteristics. 

Neon may be analyzed by GC using a thermal conductivity or a flame ionization detector. The gas may be measured by GC/MS using a capillary column. Characteristic masses for its GC/MS identification are 20 and 22. 

The application of HPLC in routine environments, like pharmaceutical, food, or environmental analysis and particularly quality assurance, makes not only great demands on the robnstness of HPLC hardware, comprising pumps, column thermostats, and detection units, bnt in addition to the column reproducibility. Column reproducibility can be investigated at different levels of complexity Run-to-run reproducibility compares consecutive chromatographic runs, whereas long-term stability describes the column variance over several hundreds of injections. Column-to-column (batch-to-batch) reproducibility finally explores the match of independently fabricated chromatographic columns. Column characteristics that are routinely consulted for the determination of the robustness are retention, selectivity, column efficiency, and peak symmetry. 

Because column performance decreases with age, it is important to keep good records of its performance. Entries useful to keep track of are chromatogram number, sample identification number, amount injected, volume injected, solvents used (A and B components), column characteristics (ID number, packing, size), back pressure, flow rate, detector setting (UV, absorbance units full scale at given wavelength), gradient conditions, results (retention time of desired product), remarks (baseline shifts, unusual wash profile, etc.), and date. 

Different kinds of HPLC exist. Many kinds of column packings and solvents are available. Retention behavior and resolution are affected by column characteristics (C-loading, chain length, porosity, etc.) and by elution scheme characteristics (mobile phase, pH, organic modifier, etc.). The samples can be separated on the basis of solubility and polarity of the sample components. 

The main advantage of the zirconium family of columns is their stability from pH 1 to 10 and at temperatures from ambient to 200°C. Their separating character also differs from silica-based columns due to the lack of ionizable surface molecules. Silica above pH 3.0 loses a proton to form anionic silicate moieties, giving the bonded-phase silica column some anionic as well as nonpolar organic column characteristics (Fig. 6.8a). 

This chapter introduces the basic theory and terminology governing chromatographic separations and the equations used to calculate the effectiveness of the analytical system. With this information, the best separation mechanism and column characteristics for a given problem can be chosen,... 

Third, the efficiency (N) may be adapted to meet the requirements set by the values of k and a. The value of N is determined by the column characteristics and the flow rate. While increasing the plate count may require great sacrifices in terms of analysis time, the reverse is also true. If the values of k and a allow the use of a column with a low N value to achieve the separation, then the analysis time may be reduced dramatically. 

In the first place column characteristics will often be determined by practical conditions, such as availability of columns and materials and instrumental considerations. 

In packed columns, there are two parameters which may be varied independently in order to optimize the column characteristics, i.e. the diameter of the column and the diameter of the particles. In open columns, only the column diameter may be varied. Additionally, the phase ratio may be varied by changing one of the capacity parameters (see section 3.5). For packed columns these parameters include the surface area of the packing material, the column porosity and the stationary phase film thickness. For open columns only the latter parameter is relevant. 

As was discussed in Chapter 1 resolution, R, is a measure of the distance between two adjacent peaks in terms of the number of average peak widths than can fit between the band (zone) centers. Assuming symmetrical (Gaussian) peaks, when R = 1, peak separation is nearly complete with only about 2% overlap. This case was shown in Chapter 1, Figure 1-4. Resolution results from the physical and chemical interactions that occur as the sample travels through the column. It should, therefore, be no surprise that resolution may also be expressed in terms of the contribution of the individual column characteristics separation factor (selectivity, a), efficiency (narrowness of peak, N), and capacity factor (residence time, k ) of the first component. The equation that describes this interrelationship is... 

Procedure Prepare a series of THI-DNPH Standard Solutions serially diluted from the Stock THI-DNPH Solution. Pipet 1, 2, and 5 mL, respectively, of the Stock THI-DNPH Solution, into separate 10-mL volumetric flasks, and dilute to volume with absolute, carbonyl-free methanol. Prepare a standard curve by injecting 5 p-L of the Stock THI-DNPH Solution, and the serially diluted THI-DNPH Standard Solutions into a 250-mm x 4-mm (id), 10-lm LiChrosorb RP-8 HPLC column (Alltech Associates, Inc., or equivalent) fitted with an ultraviolet detector set at 385 nm. The mobile phase is 50 50 (v/v) methanohO.l M phosphoric acid. Inject 5 pL of sample into the column. Adjustments in the mobile phase composition may be needed as column characteristics vary among manufacturers. At a mobile phase flow rate of 2 mL/ min and column dimensions of 250 x 4.6 mm, elute THI-DNPH at about 6.3 0.1 min. Measure the peak areas. Calculate the amount of THI in the sample from the standard curve. (For THI limits greater than 25 mg/kg, prepare a series of Standard THI-DNPH Solutions in a range encompassing the expected THI concentration in the sample.)... 

Fluid characteristics w velocity, r viscosity, p density, X thermal conductivity. Column characteristics d diameter, H height. At differential temperature between the air and the column surface. 

Figure 3. Water-column characteristics in northwestern Atlantic Ocean (38 N, 7r W August 1981).

In addition to different column characteristics, one more factor must be taken into consideration in gas chromatography, namely the sample capacity. This is defined as the maximum permissible sample size that can be injected into a column without more than 10% loss of efficiency, and it is expressed as... 

The confusion that a combination of these different definitions can generate, together with the difficulties encoimtered in the determination of some of the column characteristics involved (particularly the internal and the external porosities) makes useful a careful consideration of these issues. Given the stage of sophistication that the modeling of chromatography has now reached, it is not possible to tolerate errors, confusions, or approximations in the definitions nor in the estimations of the critical parameters related to the porosities, the velocities, and the equilibrium constants, nor to accept that more errors be made in the estimation of these parameters than those that are always involved in any measurement process. 

The countercurrent contact zone height will depend primarily upon the number of stages required ( ) and the column characteristics. The effect of backmixing also increases the column diameter. A reasonable first approximation of extraction height (L) required for agitated columns is ... 

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