|Mobile phase reservoir, filtering|
The most common type of solvent reservoir is a glass bottle. Most of the manufacturers supply these bottles with the special caps, Teflon tubing and filters to connect to the pump inlet and to the purge gas (helium) used to remove dissolved air. Helium purging and storage of the solvent under helium was found not to be sufficient for degassing of aqueous solvents. It is useful to apply a vacuum for 5-10 min. and then keep the solvent under a helium atmosphere.
High pressure pumps are needed to force solvents through packed stationary phase beds. Smaller bed particles require higher pressures. There are many advantages to using smaller particles, but they may not be essential for all separations. The most important advantages are: higher resolution, faster analyses, and increased sample load capacity. However, only the most demanding separations require these advances in significant amounts. Many separation problems can be resolved with larger particle packings that require less pressure. Thus, if the user has only moderate needs and a restricted budget, his money need not be spent on a maximum pressure pump.
Flow rate stability is another important pump feature that distinguishes pumps. Very stable flow rates are usually not essential for analytical chromatography. However, if the user plans to use his system in size exclusion mode, then he has to have a pump which provides an extremely stable flow rate. .
An additional pump feature found on the more elaborate pumps is external electronic control. Although it adds to the expense of the pump, external electronic control is a very desirable feature when automation or electronically controlled gradients are to be run. Alternatively, this becomes an undesirable feature (since it is an unnecessary expense) when using isocratic methods. The degree of flow control also varies with pump expense. More expensive pumps include such state-of-the-art technology as electronic feedback and multiheaded configurations.
Modern pumps have the following parameters:
|Flow rate range: 0.01 to 10 ml/min|
|Flow rate stability: not more than 1% (short term)|
|For SEC flow rate stability should be less than 0.2%|
|Maximum pressure: up to 5000 psi|
It is desirable to have an integrated degassing system, either helium purging, or better vacuum degassing.
Typical LC columns are 10, 15 and 25 cm in length and are fitted with extremely small diameter (3, 5 or 10 mm) particles. The internal diameter of the columns is usually 4 or 4.6 mm; this is considered the best compromise among sample capacity, mobile phase consumption, speed and resolution. However, if pure substances are to be collected (preparative scale), larger diameter columns may be needed
Packing of the column tubing with the small diameter particles requires high skill and specialized equipment. For this reason, it is generally recommended that all but the most experienced chromatographers purchase prepacked columns, since it is difficult to match the high performance of professionally packed LC columns without a large investment in time and equipment.
In general, LC columns are fairly durable and one can expect a long service life unless they are used in some manner which is intrinsically destructive, as for example, with highly acidic or basic eluents, or with continual injections of 'dirty' biological or crude samples. It is wise to inject some test mixture (under fixed conditions) into a column when new, and to retain the chromatogram. If questionable results are obtained later the test mixture can be injected again under specified conditions. The two chromatograms may be compared to establish whether or not the column is still useful.
Today, optical detectors are used most frequently in liquid chromatographic systems. These detectors pass a beam of light through the flowing column effluent as it passes through a low volume ( ~ 10 ml) flowcell. The variations in light intensity caused by UV absorption, fluorescence emission, or change in refractive index (depending on the type of detector used) from the sample components passing through the cell, are monitored as changes in the output voltage. These voltage changes are recorded on a strip chart recorder and frequently are fed into an integrator or computer to provide retention time and peak area data.
The most commonly used detector in LC is the ultraviolet absorption detector. A variable wavelength detector of this type, capable of monitoring from 190 to 460-600 nm, will be found suitable for the detection of the majority samples.
Other detectors in common use include: refractive index (RI), fluorescence (FL), electrochemical (EC) and mass-spectrometric (MS). The RI detector is universal but also the less sensitive one. FL and EC detectors are quite sensitive (up to 10-15 mole) but also quite selective. The MS detector is the most powerful one but it still the most complicated and most expensive.
Sample introduction can be accomplished in various ways. The simplest method is to use an injection valve. In more sophisticated LC systems, automatic sampling devices are incorporated where sample introduction is done with the help of autosamplers and microprocessors.
In liquid chromatography, liquid samples may be injected directly and solid samples need only be dissolved in an appropriate solvent. The solvent need not be the mobile phase, but frequently it is judiciously chosen to avoid detector interference, column/component interference, loss in efficiency or all of these. It is always best to remove particles from the sample by filtering, or centrifuging since continuous injections of particulate material will eventually cause blockage of injection devices or columns.
Sample sizes may vary widely. The availability of highly sensitive detectors frequently allows use of the small samples which yield the highest column performance. Typical sample mass with 4.6 mm ID columns range from the nanogram level up to about 2 mg diluted in 20 ml of solvent. In general, it will be noted that much less sample preparation is required in LC than in GC since unwanted or interfering compounds, or both, may often be extracted, or eliminated, by selective detection.
Since the detector signal is electronic, use of modern data acquisition techniques can aid in the signal analysis. In addition, some systems can store data in a retrievable form for highly sophisticated computer analysis at a later time.
The main goal in using electronic data systems is to increase analysis accuracy and precision, while reducing operator attention. There are several types of data systems, each differing in terms of available features. In routine analysis, where no automation (in terms of data management or process control) is needed, a pre-programmed computing integrator may be sufficient. If higher control levels are desired, a more intelligent device is necessary, such as a data station or minicomputer. The advantages of intelligent processors in chromatographs are found in several areas. First, additional automation options become easier to implement. Secondly, complex data analysis becomes more feasible. These analysis options include such features as run parameter optimization and deconvolution (i.e. resolution) of overlapping peaks. Finally, software safeguards can be designed to reduce accidental misuse of the system. For example, the controller can be set to limit the rate of solvent switching. This acts to extend column life by reducing thermal and chemical shocks. In general, these stand-alone, user programmable systems are becoming less expensive and increasingly practical.
Other more advanced features can also be applied to a chromatographic system. These
features include computer controlled automatic injectors, multi-pump gradient controllers
and sample fraction collectors. These added features are not found on many systems, but
they do exist, and can save much time and effort for the chromatographer.