Guard columns. HPLC

An HPLC typically includes two columns an analytical column responsible for the separation and a guard column. The guard column is placed before the analytical column, protecting it from contamination. 

Finally, the useful life of an analytical column is increased by introducing a guard column. This is a short column which is placed between the injector and the HPLC column to protect the latter from damage or loss of efficiency caused by particulate matter or strongly adsorbed substances in samples or solvents. It may also be used to saturate the eluting solvent with soluble stationary phase [see Section 8.2(2)]. Guard columns may be packed with microparticulate stationary phases or with porous-layer beads the latter are cheaper and easier to pack than the microparticulates, but have lower capacities and therefore require changing more frequently. 

In SEC analysis of additive extracts from polymers, the effect of the extraction solvent on the mobile phase is less critical than in HPLC analysis. The extraction solvents typically employed generally do not interfere with the SEC mobile phases. Moreover, the same solvents are often used both as extraction solvent and as mobile phase. Therefore, there is no need to evaporate the extract to dryness prior to analysis and then to redissolve it in a suitable solvent. Typical extraction procedures often produce extracts that generally contain a small amount of wax. Frequently, removal of such oligomers from an extract is necessary, e.g. by means of precipitation, centrifuging, precolumn filtration or protection (use of a reversed-phase guard column). In SEC separations the presence of polyolefin wax does not usually disturb provided that the MW of the wax is higher than that of the analysed compounds. 

High Performance Liquid Chromatographic (HPLC) Analysis. A Waters HPLC system (two Waters 501 pumps, automated gradient controller, 712 WISP, and 745 Data module) with a Shimadzu RF-535 fluorescence detector or a Waters 484 UV detector, and a 0.5 pm filter and a Rainin 30 x 4.6 mm Spheri-5 RP-18 guard column followed by a Waters 30 x 3.9 cm (10 pm particle size) p-Bondapak C18 column was used. The mobile phase consisted of a 45% aqueous solution (composed of 0.25% triethylamine, 0.9% phosphoric acid, and 0.01% sodium octyl sulfate) and 55% methanol for prazosin analysis or 40% aqueous solution and 60% methanol for naltrexone. The flow rate was 1.0 mL/min. Prazosin was measured by a fluorescence detector at 384 nm after excitation at 340 nm (8) and in vitro release samples of naltrexone were analyzed by UV detection at 254 nm. 

FIGURE 2.1 Flow diagram for dual-stream HPLC with guard column regeneration. 

FIGURE 2.3 Autosampler set-up for performing dual-stream HPLC with guard column regeneration (A) overall view, (B) zoomed view of the valving configuration. 

HPLC condition — A Waters reversed-phase HPLC column (Symmetry Shield RP Cl8, 5 pm, 2.1 x 50 mm) was used in conjunction with a Regis SPS guard column (ODS, 5 pm, 100 A,... 

Fogli et al. developed and validated an HPLC method with fluorescence detection for simultaneous routine TDM of anthracyclines and their metabolites.27 They coupled a Waters LC Module I Plus system equipped with a WISP 416 autosampler with a Model 474 scanning fluorescence spectrophotometer. The stationary phase was a Supelcosil LC-CN column (250 x 4.6 mm, 5 /um particle size) with a /iBondapak-CN guard column. The mobile phase consisted of 50mM monobasic sodium phosphate buffer and acetonitrile (65 35 v/v), adjusted to pH 4.0 with phosphoric acid. The flow rate was 1 mL/min. The fluorescence detection was set at excitation wavelengths of 233, 254, and 480 nm and at an emission wavelength of 560 nm. 

A guard column is a short, less-expensive liquid chromatography column that is placed ahead of the analytical column in an HPLC system. The purpose of a guard column is to adsorb and retain mixture components that would contaminate the more expensive analytical column. In-line filters are relatively coarse filters (compared to prefilters) placed in the mobile phase line to filter out particulates that maybe introduced on-line, such as from sample injection. 

In addition to the commercially available systems, several authors have described laboratory-built systems using commercially available components from companies such as Upchurch Scientific (Oak Harbor, WA). One of the first reported laboratory-built micro-bore HPLC systems was described by Simpson and Brown, which was a simple adaptation of a standard HPLC system to accept micro-bore columns built from guard columns. A complete system has been described based on dual microdialysis syringe pumps (CMA Microdialysis, Chelmsford, M A) or dual syringe pumps (Harvard Apparatus, Inc., Holliston, MA), a microinjection port, and a micro-column the latter components being obtained from Upchurch scientific (Figure 3.5). This system was coupled with a laser-induced fluorescence (LIF) detector and used to measure neuropeptides in sub-microliter samples. A further modification of this system was built to perform immunoaffinity isolations of biomedically important analytes from clinical samples. ... 

In the previously described approaches, different HPLC pumps are used in the two dimensions. A different approach, where the flow from one single pump was splitted and introduced to one first- and two second-dimension columns, was used by Venkatramani and coworkers [45,47]. In this approach, a 12-port valve equipped with three loops [45] or with three guard columns [47] was used as an interface (Figure 4.8). 

---