Column frits

At the two lower concentrations, the orange color of methyl orange was confined to the top of the GPG spin column and with increasing concentrations the orange color moved down the column towards the top of the spin column frit, consistent with the ESI-MS intensity observations. Similar dose response titration studies have been reported with stronger binders, e.g., matrix metalloproteinase-1 (MMP-1) protein with a substituted hydroxyamide WY252 with an IC50 of 9.9 pM (see Fig. 2.7) [15]. 

The high viscosity of some high MW samples is known to cause flow rate upsets as the sample passes through the SEC column frits. Such flow rate upsets often occur at the time of elution of the sample. While the flow rate upsets like this are likely to cause viscosity detection errors in most other SEC viscosity detectors, the signal of the present viscosity detector, however, will remain true, cind unaffected by the high sample viscosity problem. 

Dispersion in column frits was originally thought to be large and thus, made a significant contribution to the overall extra column variance. It was not until the introduction of low-dispersion unions that it was found that most of the dispersion that was thought to occur in the frits, actually occurred in the unions that contained the frits. Scott and Simpson (11) measured the dispersion that occurred in some commercially available column frits and demonstrated that their contribution to dispersion to be insignificant compared with other sources of extra column dispersion. 

Samples, even at moderate concentrations, injected into the HPLC column may precipitate in the mobile phase or at the column frit. In addition, the presence of other compounds (e.g., lipids) in the injection sample may drive the carotenoids out of solution or precipitate themselves in the mobile phase, trapping carotenoids. It is best to dissolve the sample in the mobile phase or a slightly weaker solvent to avoid these problems. Centrifugation or filtration of the samples prior to injection will prevent the introduction of particles that may block the frit, fouling the column and resulting in elevated column pressure. In addition to precipitation, other sources of on-column losses of carotenoids include nonspecific adsorption and oxidation. These can be minimized by incorporating modifiers into the mobile phase (Epler et al., 1993). Triethylamine or diisopropyl ethylamine at 0.1% (v/v) and ammonium acetate at 5 to 50 mM has been successful for this purpose. Since ammonium acetate is poorly soluble in acetonitrile, it should be dissolved in the alcoholic component of the mobile phase prior to mixing with other components. The ammonium acetate concentration in mobile phases composed primarily of acetonitrile must be mixed at lower concentration to avoid precipitation. In some cases, stainless steel frits have been reported to cause oxidative losses of carotenoids (Epler et al., 1992). When available, columns should be obtained with biocompatible frits such as titanium, Hastolloy C, or PEEK. 

Owing to reported losses of carotenoids by rapid decomposition through oxidation in the presence of a stainless steel column frit (158), it has been recommended to purchase HPLC columns fitted with inert metal-free frits (159,160). 

It s better to pick a time convenient for you than to have to do this process on an emergency basis in the middle of a critical separation. I would have a tested column ready as a replacement. Replace the dirty column after washing out the buffer, cap it, and, then, wash the old column off-line when you have more time. You never seem to wash everything off the column. After you ve used a column for a while, you often will find a brown or black residue at the column head under the column frit on opening even a freshly washed column. Don t worry about it if the column standards run correctly. 

Treatment Replace outlet column frit. Sonicate old frit with 10% NaOH, water, and again with water (see 3b). 

Another approach to fraction collection is the use of an on-column frit structure or capillary fracture that depends on the electroosmotic flow to deposit the eluent in a continuous manner on a moving surface. Although this approach circumvents the dilution problem, the collection structures are complex and can result in the loss of some of the analyte. One commercially available fraction collection device couples CE with membrane fraction collection, without the need for frits or capillary fractures. The outlet vial holder can be removed and replaced with a wetted circular polyvinylidene difluoride (PVDF) disk, which enables the collection of eluted analytes and subsequent manipulations such as immunoblotting and microsequencing. Figure 6.13 shows a schematic diagram of the CE membrane fraction collector interface.74... 

Band tailing causes inferior resolution and reduced precision. Thus conditions resulting in tailing or asymmetric peaks should be avoided. Peak asymmetry or band tailing can arise from several sources partially plugged column frits, void(s) in the column, buildup of sample components and impurities on the column inlet following multiple sample injections, sample overload, solvent mismatch with reference to the sample, chemical or nonspecific interactions (e.g., silanol effects), contamination by heavy metals, and excess void volume in the HPLC system. 

A clogged column frit is another common HPLC problem. To minimize this problem from the start, the... 

Flush the empty column with the slurry solvent prior to use to ensure that the column, column frits, and plumbing are clean and wetted. Empty the column before introducing the slurry suspension. 

Many different types are available from numerous manufacturers. Different brands must not be mixed They often differ in the angle of the ferrule and in their stop depth, from 0.1700 in = 4.32 mm (Rheodyne) to 0.0800 in = 2.03 mm (Valeo). Usually the thread is English not metric but the length of the thread is also not identical with all brands. If the stop depth is less than it should be there is an extra-column volume in the system if it is too long the capillary will be damaged or the column frit will be pushed into the chromatographic bed. 

This reduced flow resistance is much too high. The column, frit, or a part of the instrument is clogged. 

Filtration is used as a sample-preparation method to remove particulates and debris that can potentially foul the LC lines, column frits, or mass spectrometer interface. Also, it is generally accepted that all workstations and pipetting systems can beneht from sample hltration because of the universal issues related to plasma clot formation that introduce pipetting challenges. Applications that use hltration include the removal of a mass of precipitated protein or of debris from raw plasma before use with any of the traditional sample-preparation techniques, as well as direct injection techniques (turbulent how... 

There are two methods of adsorbing compounds on the lon-exchange resin batch mode or column mode. In batch mode, an appropriate amount of resin IS added to a vessel containing the clarified broth. The mixture is then stirred for an appropriate amount of time, typically 15-30 min, and the resin is filtered. Such a batch or slurry mode of operation is advantageous in cases in which the broth cannot be easily filtered, a state that will rapidly block column frits. The resin can be recovered by filtration through a very coarse medium such as a fine wire screen or sometimes simply by decanting the supernatant spent broth. 

---