Columns inner diameter and

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. 

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 ... 

Column dimensions—length (L) and column inner diameter (dc or i.d.)— control column performance (N, speed, sensitivity, sample capacity) and its operating characteristics (flow rate, back pressure). Designations of various column types based on column inner diameters and their associated characteristics are shown in Table 3.1. Note that void volume, sample capacity, and operating flow rate are proportional to (dc)2, while detection limit, or sensitivity, is inversely proportional to (dc)2. Note also that prep columns (>10mm i.d.), microbore (micro columns (<0.5 mm i.d.) will require specialized HPLC instruments (see Chapter 4). There is a definitive trend toward the increased use of shorter and smaller inner diameter analytical columns due to their higher sensitivity performance and lower solvent usage.9"11 This trend will be explored later. 

TABLE 3.11 Selected Phase Ratios as a Function of Column Inner Diameter and Film Thickness... 

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). 

Note not all combinations of stationary phase, column internal diameter, and column length are available. For analytical comparisons, see References 36 and 37. Names of columns and/or stationary phases may be trademark protected. N/A = not available. ID = inner diameter. 

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. 

The use of miniaturized systems might provide a feasible approach for speeding up the separations (given that smaller column dimensions, in terms of both the inner diameter and the length, decrease the dilution). However, miniaturization is not necessarily synonymous with fast separations, since problems often arise with dead volumes, caused by the connections. Nano-LC has been used with UV or MS detection for the analysis of atenolol in urine [143]. A homemade column with an internal diameter of 75 jm containing diol silica modified with teicoplanin was used as the CSR... 

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.]...

Column volume. X. The total volume of the column which contains the stationary phase. (IUPAC recommends the column dimensions be given as the inner diameter and the height or length of the column occupied by the stationary phase under the specific chromatographic conditions. Dimensions should be given in millimeters or centimeters.)... 

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. 

Liquid chromatography (LC) is the most commonly used technique for trace element speciation with ICP-MS detection. The mobile phase flow rates used with most LC techniques (0.5-2.0 mL min-1) are compatible for ICP-MS introduction using conventional sample introduction systems (pneumatic nebulization with cross flow and concentric nebulizers and double-pass spray chambers). An interface, known as a transfer line, must be constructed to allow connection between the outlet of the LC column and the nebulizer of the ICP-MS. Inert plastic tubing is commonly used for this purpose with the inner diameter and length kept to 20-50 cm in order to minimize peak broadening. 

Figure 7 CEC chromatogram with ultrashort packed capillary column. Column 75 pm in inner diameter and 15 mm in length pcked with IC-CATION-SW. Eluent 30% methanol aqueous solution containing 30 mM KH2P04 and 25 mM EGTA. Aplied voltage, -3.0 kV. Injection, -3.0 kV for 0.5 s. Samples (a) uracil (b) adenine (c) cytosine (d) dopamine (e) serotonin.

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%. 

Figure 2 shows a schematic drawing of how the SDS-removal column is attached to the reversed phase column. To prepare this column setup, a fused silica tubing with the desired dimensions (360 im outer, 250 )xm inner diameter and 200 mm length for LC-MS analysis) was connected to a transfer tubing (180 im outer and 50 pm inner diameter) holding a frit in place. The column was filled with reversed phase support as described in... 

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]. 

Fiuther developments of nano-LC comprise further reduction of coltunn inner diameter and the use of smaller particles to enhance the separation efficiency. Some examples of these developments are the use of a 150-pm-ID in-needle coltunn with a 0.3-0.5-pm-ID laser-pulled emitter tip, packed with 1-pm particles, and operated at flow-rates of <50 nl/min [60], and the ttse of long 15-75-pm-ID nano-LC columns, packed with 3-pm particles, and operated at flow-rates as low as -20 nl/min [54]. 

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