Pumps size exclusion chromatography

The instrumentation of HdC, including a pump, an injector, a column (set), a detector, and a recorder or computer, is very similar to size exclusion chromatography SEC). The essence of this technique is the column. There are two types of HdC columns open microcapillary tubes and a nonporous gel-packed column. This chapter emphasizes column technology and selection and the applications of this technique on the molecular weight analysis of macromolecules. 

Novolac molecular weights were measured in THF at 35°C by high pressure size exclusion chromatography using a Waters Model 510 pump (flow rate=1.0 ml/min), 401 differential viscometer detector and a set of Dupont PSM 60 silanized columns. A universal calibration curve was obtained with a kit of 10 narrow molecular weight distribution, linear polystyrene standards from Toya Soda Company. Data acquisition and analysis were performed on an AT T 6312 computer using ASYST Unical 3.02 software supplied with the Viscotek instrument. 

Instrumentation. A Pharmacia BioPilot Column Chromatography system was used to perform large-scale size exclusion chromatography (SEC) with an 11.3 x 90 cm BioProcess column packed with Sephacryl S-200 HR gel. High performance size exclusion (HPSEC) and ion exchange chromatography (HPIEC) were conducted with Pharmacia Superose 6 and 12 (HR 10/30) and Mono-Q (HR 5/5) columns respectively, equipped with Beckman model 520 system controller and Beckman model HOB HPLC pumps. 

The constant-flow pumps is the most widely used in all common IC applications. Flow rate stability is an important pump feature that distinguishes pumps. For size exclusion chromatography, the flow rate has to be extremely stable. External electronic control is a very desirable feature when automation or electronically controlled gradients are to be run. 

IR, UV, and H NMR spectra were obtained using Jasco FTIR-410, Shimadzu UV-2400PC, and Jeol JNM-GX270 (270 MHz) spectrometers, respectively. Size exclusion chromatography (SEC) was carried out on a Jasco GPC equipment consisting of a PU-980 pump, an RI-930, and a Shodex KF-806M column with polystyrene as a standard and tetrahydrofliran (THF) as an... 

Size exclusion chromatography, also called gel permeation chromatography (GPC) continually pumps solvent through a series of columns filled... 

The determination of molecular mass and molecular mass distribution by size-exclusion chromatography is rapid and easy. However, as it is based exclusively on elution volume, this latter must be determined extremely accurately. Elution volume is a function of log(molar mass) and hence even small errors in the determination of Ve have a dramatic effect. For this reason, either a pump which is both reproducible and precise plus thermostatic control of the system should be... 

The high salt concentrations used can present a challenge for the HPLC equipment. Pumps with seal-wash are prrferred. If chloride buffers are used, HPLC systems with nonmetallic fluid paths are recommended. It is also advisable to flush the salt solution out of the system when it is not in use. Temperature and pH can affect the separation and should therdbre be controlled. But both the temperature effect and pH effect are smaller than in other forms of chromatography (with the exception of size-exclusion chromatography). 

Pumps are usually constructed from stainless steel, check valves and pistons from ruby or sapphire, and the seals from PTFE. These are the only materials which come into contact with the solvent and are inert to most of the common solvents. Unstabilized chloroform can present problems when used with inferior grades of stainless steel due to attack by hydrogen chloride generated from decomposition of the chloroform. In some types of chromatography, low concentrations of metal ions extracted from the pump components by the solvent can cause interference with the analyses. For these applications it is possible to obtain pumps constructed with ceramic or titanium heads. However, these problems do not normally arise in size exclusion chromatography. 

Figure 2.2 Typical setup of size-exclusion chromatography equipped with triple detection for determination of the moiecuiar weight of polymers without the need for prerequisite calibration. DRi, differential refractive index detector DV, differentiai viscometer I, injector MALS, multi-angle light-scattering detector (depending on the model, the scattered light is measured at 3 or 18 different angles) P, pump. The central unit includes a computer and software.

Many components of SEC systems are well known and ubiquitous in an analytical lab. Size exclusion chromatography systems require at least an isoaatic pump, a high-pressure injection systan (manual or automated), one or more SEC colunm(s), one or more detector(s), and data evaluation units with software to acquire, calibrate, and analyze data. Other components, such as degassers or column compartments are often optional and depend on the particular application and on the lab environment and conditions. Although SEC and high-performance liquid chromatography (HPLC) seem to be very similar in instrumentation requirements, the theoretical background and separation mechanism of both techniques are entirely different. The major differences in the practice of SEC are outlined in Table 9.2. 

Equipment for running size-exclusion chromatography. The buffer is pumped through the column (right) by a computer-controlled device... 

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