Columns for HPLC

Most HPLC columns are made of 316 grade stainless steel, which is austenitic chromium-nickel-molybdenum steel, USA standard AISI, resistant to the usual HPLC pressure and also relatively inert to chemical corrosion (chloride ions and lithium ions at low pH being important exceptions). The inside of the column should have no rough surfaces, grooves or microporous structures, so the steel mbes must be either precision drilled or polished or electropolished after common manufacturing, e.g. by drawing. 

Glass tubes are smooth, chemically inert (they do not alter the sample substances at all) and do not corrode. For HPLC they are used as glass-lined steel tubes which are pressure-resistant. 

Tantalum is rarely used. It is less prone to corrosion than steel and its surface is smooth, but it does not give, so that fittings have to be glued rather than pressed on. It is very expensive. 

the pressure-resistant plastic mentioned in Section 4.4, cannot only be used for capillaries but also for column tubing. 

Flexible polyethylene tubes compressed by a hydraulic fluid in a suitable casing represent a special type of column system (Waters). The column wall adapts to suit the packing, thus preventing wall effects and the formation of channels. 

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Analytical Columns The most commonly used columns for HPLC are constructed from stainless steel with internal diameters between 2.1 mm and 4.6 mm, and... 

Shodex Packed Columns for HPLC and Shodex Instruments for HPLC, 95/96, p. 10, Showa Denko. 

A second approach to on-line SPE is to use an SPE extraction column that can be used for hundreds of samples. In the simplest of systems, two pumps (either HPLC systems or stand-alone pumps) are connected to an extraction column and an analytical column via 6 or 10 ports, and these are further linked to an MS system. The pump that is connected in-line with the autosampler loads the sample under high flow rate (3 to 5mL/min). The large molecules from the matrix are not retained by the SPE sorbent and are diverted to waste. The analytes of interest are retained by the sorbent. The valve then switches so that the second pump with the elution solvent is now in-line with the SPE column and elutes the analytes onto the analytical column for HPLC/MS analysis. This type of system has proved useful for the analysis of small molecules in a variety of sample matrices such as plasma and urine. While it is relatively straightforward to plumb this type of system with components already in the laboratory, commercial systems are available from such companies... 

In the second approach non-procedure related factors are considered. Factors such as e.g. different laboratories, different analysts, different instruments, different lots of reagents, different days, different columns for HPLC methods or different plates for TLC methods are then examined. 

TABLE 16. Column COLUMNS FOR HPLC -2 Mobile Phase Application... 

C18 reversed-phase column for HPLC separations of chlorophylls narrow-bore (2.1 -mm i.d.) column at flow rate of 50 to 300 p.l/min without splitting the flow, or analytical column (4.6-mm i.d.) at 1 ml/min with post-column solvent splitting of 5 1 (200 p,l/min entering the mass spectrometer) for APCI electrospray interfaces are available for use without solvent splitting over all flow rates from nl/min to 1 ml/min... 

As stated in Basic Protocol 1, the method of HPLC used may change depending on the organic acids present in the sample. This method uses a Bio-Rad Aminex HPX-87H column for HPLC analysis. An internal standard is important for the analysis of organic acids. This provides a means of not only determining if the analysis is working but also quantitating the percent recovery of the method. 

Table 4 Survey of Commercially Available Reversed-Phase Prepacked Columns for HPLC of Proteins11...

D Johns. Columns for HPLC separations of macromolecules. In RWA Oliver, ed. HPLC of Macromolecules, a Practical Approach. Oxford IRL Press, 1989, pp 1-42. 

Recent advances in chromatography have made it possible to employ microbore HPLC for the determination of NOC. Its main advantage is that it uses a very low mobile-phase flow (20-100 /rl/min). This might make the TEA compatible with a reversed-phase system. Massey et al. (73), in fact, have successfully used reversed-phase chromatography for the HPLC-TEA determination of V-nitroso-V, 7V -di methylpiperazinium iodide. A 500-mm X 1-mm microbore ODS column and a mobile phase consisting of 0.1 M ammonium heptane-sulfonate in methanol water (70 30) (flow rate 20 /zl/min) was used for the HPLC separation. In another study, Riihl and Reusch (74) used a microbore Spherisorb 3 SW column for HPLC-TEA determination of volatile V-nitrosamines. The mobile phase was a mixture of 2-propanol and n-hexane (2.5 97.5). Further application of such techniques for the determination of various polar NOC, especially A-nitrosamides, in foods is desirable. 

The two main methods of packing columns for HPLC are dry packing, which is suitable for particles of diameter > 30 jim, and balanced-density slurry packing which is best for small particles of diameter 5-30 jum. 

Aqueous samples or aqueous extracts of nonaqueous samples directly injected onto a C-18 reverse phase column for HPLC analysis with UV detection at 195 nm (U.S. EPA Method 8316, 1992) mobile phase, water flow rate 2 mL/min pressure 38 atm. 

This technique is now used extensively to assess enantiomeric excesses in organic reactions and separate small quantities of enantiomers. Closely related chiral corands are particularly useful in assessing the optical purity of amino acids, although modern chiral columns for HPLC may cost in excess of US 2000. 

Small-bore columns for HPLC are categorized in terms of the column internal diameter. There are four types of small-bore columns narrow-... 

Johns, D. (1989). Columns for HPLC separation of macromolecules. In HPLC of Macromolecules A Practical Approach (R. W. A. Oliver, ed.), pp. 1-42. Oxford University Press, Oxford. 

In the following, the synthesis of the most often employed stationary phase is discussed spherical silica with an n-octadecyl modification. The synthesis route has been chosen because all synthesis steps are well characterized and documented in standard operation procedure (SOP) protocols. The objective of this work was to develop a manufacturing process for a reversed phase C18-bonded silica column for HPLC according to standardized and validated procedures and to perform certification of the column, the tests and the mobile phases (du Fresne von Hohenesche et al., 2004). Figure 3.13 shows a scheme of the whole manufacturing process, and Table 3.7 summarizes the main steps. 

Reverse-phase (RP) methods were initially established on the basis of similarity between octanol water partitioning and retention on an RP column for HPLC... 

Tanaka, N. Nagayama, H. Kobayashi, H. Ikegami, T Hosoya, K. Ishizuka, N. Minakuchi, H. Nakanishi, K. Cabrera, K. Lubda, D. Monolithic Silica Columns for HPLC, micro-HPLC and CEC, J. High Resol. Chromatogr. 23, 111-116(2000). 

Generally, the expert chromatographer relies on his own column packing techniques, because comnercial packed columns for HPLC do not always provide optimum efficiency, but are less vulnerable to the treatment of less experienced chromatographers. 

Figure 3.6 Standard column and guard column for HPLC. View of the ZORBAX column assembly and its exploded view. The replaceable precolumn prevents clogging of the analytical column while extending its lifetime and preserving its performance (Reproduced courtesy of RTI).

How do the capacities of columns for HPLC compare to those for ion chromatography ... 

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