Column inner diameter

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). 

Many precolumns and trap cartridges for sample clean-up are commercially available. In our experience, a 2 to 3 cm short column with twice the analytical column inner diameter and packed with the same particles performs satisfactorily. An antibody affinity column for selective removal of highly abundant proteins from human serum samples provides better sensitivity for the discovery of low abundance protein markers that may represent revolutionary therapeutic diagnosis and monitoring. 

Equipment, column inner diameter (cm) Elow rate (Eh) Amount stationary phase (kg) Throughput, feed (g/day)... 

Column inner diameter (mm) Flow rate (ml/min) Loading (mg)... 

Micro-HPLC operation sets special demands on the gradient instrumentation. As the internal column diameter, d, decreases, lower flow rates should be used at comparable mean linear mobile phase velocities, u = 0.2-0.3 mm/s. At a constant operating pressure, the flow rate decreases proportionally to the second power of the column inner diameter, so that narrow-bore LC columns with 1mm i.d. require flow rates in the range of 30-100pL/min, micro-columns with i.d. 0.3-0.5mm, flow rates in between 1 and lOpL/min, and columns with 0.075-0.1 mm i.d. flow rates in the range of hundreds nL/min. Special miniaturized pump systems are required to deliver accurately mobile phase at very low flow rates in isocratic LC. 

Purification by pressure column chromatography on silicagel is conducted as follows. A glass column (inner diameter 5 cm, length 51 cm) is filled with a slurry of silica (450 g of silicagel, Acros, 0.035-0.070 mm, pore diameter 6 nm) in 95 5 (v v) CH2Cl2/acetone. A 3-g portion of cmde [AsPl JL is dissolved in a minimum amount of solvent and adsorbed on the top of the column. The column is connected with a membrane pump (output pressure 1.6 bar) and then eluted at a flow rate of about 180 mL/min. The violet fraction is collected and evaporated on a rotary evaporator. This chromatographic separation on the whole lasts 8 h. 

Figure 24-4 Effect of open tubular column inner diameter on resolution. Narrower columns provide higher resolution. Notice the increased resolution of peaks 1 and 2 in the narrow column. Conditions DB-1 stationary phase (0.25 xm thick) in 15-m wall-coated column operated at 95°C with He linear velocity of 34 cm/s. [Courtesy JSW Scientific. Folsom. CA.]...

For strong UV absorbers such as isoflavones, total injected amounts of <20 pg and injection volumes of 10 to 30pi are typical. Larger injection volumes can cause broadening of peaks. The flow rate was developed based on the column inner diameter and mass sensitivity. 

The correlation between analytical and preparative procedures is found by calculating the square of the ratio of analytical to preparative column inner diameters and multiplying that value by the flow rate of the analytical system ... 

Reducing the column diameter from 4.6 to 2 mm, results in a theoretical fivefold increase in peak concentration. This means that the injected sample is approximately 20% less diluted and an increase in detectability can be expected. The increased peak concentration obtainable by decreasing the column inner diameter, with all other parameters equal, is balanced by the decreased sample volume that should theoretically be injected. The only way to see the increased peak concentration, under these conditions, is to concentrate a large sample volume on the head of the column prior to running the separation. 

The column inner diameters for the four classes of small bore columns are... 

As an example, a conventional HPLC column of 20 cm length packed with 5 pm (0.005 mm) particles yields 10,000 plates (column III in table 7.1). With the conventional column inner diameter of 5 mm we find... 

P. Molander, R. Olsen, E. Lundanes, and T. Greibrokk,The impact of column inner diameter on chromatographic performance in temperature gradient liquid chromatography, AzzaZysf 128 (2003), 1341-1345. 

The Chitopearr resin was packed in a glass column (inner diameter 1. 4 cm, bed height 4.3 cm). The column was first equilibrated with a 20 mM MES buffer containing 20 mM NaCI and 10 mM 2-mercaptoethanol. Then 60 ml of the MES buffer containing metal ion was applied at the flow rate of O.S ml/min. Adsorption capabilities of the ligands were examined for cadmium, gallium, cupric, zinc, or nickel ion. After the column was washed with MES buffer, the adsorbed metal ion was eluted with the MES buffer (pH of which was adjusted to pH 2.0). In order to examine effects of pH on the adsorption, pH of the MES buffer was varied from pH S to pH 9. The eluted solution was collected as several fractions of 10 ml each, and the metal concentration of each fraction was determined with atomic adsorption analysis (SAS 7S00A, Seiko Instruments, Japan). Total amount of the eluted metal ion was defined as the adsorbed metal ion on the resin. The total amount of the adsorbed metal ion was divided by the total amount of immobilized protein to calculate the number of metal molecules bound to one mole of the protein. The adsorption experiments were carried out multiple times, and the maximum experimental error was 25%. 

The coltrrrm is the heart of the LC system. It requires appropriate care. Conventionally, LC colurtms are 100-300-mm long and have an internal diameter of 3. 6 mm with an outer diameter of V4 inch. In LC-MS, and especially in qrrantitative bioanalysis, shorter colurtm are used, e.g., 30-50 mm, and packed with 3-5 pm ID packing materials. A variety of other colttmn types, differing in column inner diameter, are apphed. Some characteristics of these colttrtms are compared in Table 1.1. 

From a practical point of view, the discussion on flow-rate can be summarized as follows. In LC-APCI-MS, the typical flow-rate is 0.5-1.0 ml/min. For routine applications of LC-ESI-MS in many fields, extreme column miniaturization comes with great difficulties in sample handling and instrument operation. In these applications, LC-MS is best performed with a 2-mm-ID column, providing an optimum flow-rate of 200 pFmin, or alternatively with conventional 3-4.6-mm-ID columns in combination with a moderate split. In sample limited cases, further reduction of the column inner diameter must be considered. Packed microcapillary and nano-LC columns with micro-ESI and nano-ESI are rontinely applied inproteomics stndies (Ch. 17.5.2). 

The column inner diameter is determined by the amount of sample available and the LC-MS interface selected, tn general, flow-rates between 200 and 400 pl/min are considered optimum for (pneumatically-assisted) ESt. This explains the frequent use of 2-mm-ID columns, tn sample-limited analysis, e.g., in the analysis of mouse plasma samples, microbore (1 mm ID) or packed-microcapillary columns (320 pm ID) are applied at relatively low flow-rates [12-13]. For APCt, 4.6-mm-tD columns are preferred, operated at typically 1 ml/min. The LC system should provide symmetric peaks with a width that enables the acquisition of tO-20 data points for each compound in order to enable an accurate determination of the peak area. 

The mono-, di-, and tri-phosphorylates of d4T were analysed by ion-pair LC-MS after lysis of the PBMC cell in Tris/methanol and centrifugation [50]. The supernatant was injected into the LC system with a 150x2.1-mm-ID Cjg column (5 pm) and a mobile-phase gradient of 70 to 35% solvent A (10 mmol/1 DMHA and 3 mmol/1 ammonium formate adjusted at pH 11.5) in solvent B (50% acetonitrile in 20 mmol/1 DMHA and 6 mmol/1 ammonium formate). Negative-ion ESI-MS was performed in SRM mode. The method enabled the direct measurement of the chain terminator ratio (d4T-triphosphate/deoxythymidine-triphosphate). Subsequently, the same group [51] reported modifications of this method, including simplifications of the sample pretreatment, replacement of the LC column for another type, and reduction of the column inner diameter from 2 mm ID to 0.32 mm ID. This improved method was applied to the determination of the phosphorylates of d4T, 3TC, and ddl. The sample throughput is 200 samples per week. The determination of intracellular AZT-triphosphate in PBMC [52], and the validation of the method for the determination of the ddl and d4T triphosphates was reported separately [53]. 

Because the column inner diameter is small, the amount of stationary phase is also very small and, as a consequence, the amount of sample that can be introduced into the column without overloading is very small (typically a few nanoliters). In most cases, the preferred sample introduction system consists of an injection valve containing an internal loop smaller than 0.1 ijuh. 

A compact sensor of greatly reduced dimensions (outer diameter x length 36 x 46 mm) has been constructed and is shown in Fig. 2. In order to conveniently accommodate enzyme columns and to ensure isolation from ambient temperature fluctuations, a cylindrical copper heat sink was included. An outer Delrin jacket further improved the insulation. The enzyme column (inner diameter x length 3x4 mm), constructed of Delrin, was held tightly against the inner terminals of the copper core. Short pieces of well-insulated gold capillaries (outer diameter/inner diameter 0.3/0.2 mm) were placed next to the enzyme column as temperature-sensitive elements. Microbead thermistors were mounted on the capillaries with a heat-conducting epoxy. Two types of mini system has been constructed as discussed below. 

The lowermost graphs show linear relationships. The flow rate is proportional to the linear flow velocity (lower left). It depends from the porosity and the column inner diameter. Again, s = 0.65 for bonded phases. With s = 0.8, typical for bare silica, the lines would be steeper. The graph shows the data for three different column diameters. 

Figure 24.1 Peak shapes as obtained with different column diameters and separation performances (as a function of particle diameter at a given column length). Columns 1 and 3 are packed with a coarse stationary phase, columns 2 and 4 with a fine one. The packing quality, defined as reduced plate height h, is the same in all four cases. Separation performance is independent of column inner diameter therefore peaks 1 and 3 as well as 2 and 4, respectively, are ofthe same width. The peak height in the eluate can be calculated from equation (15) it is higher when the column is thinner and the separation performance is better. Peak areas cannot be compared although the same amount is injected in any case if a concentration-sensitive detector is used optimum flow rate depends on particle size and hence also the residence time in the detector.

In contrast to the concentration, the minimum detectable mass depends on the retention factor of the solute. The earlier a peak is eluted, the smaller is the maximum injection volume and, with the concentration of the sample solution being constant, the smaller is the absolute mass of solute injected. The same is true if the column inner diameter is reduced. 

Typical capillary column GC curves can be similarly generated, as shown In Figure 1c. In this case an Intermediate value for the binary diffusivity, 0.2 cm /s, was used along with two column Inner diameters, 250 pm and 50 pm. The 50 pm diameter Is unusually small for ultrahlgh resolution capillary GC work while the 250 pm diameter Is in the middle of the diameter range commonly used in this area. As seen in Figure 1c, a comparison of the 50 pm diameter SFC curve to the 50 pm GC curve (Curves 3 and... 

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