Modern High Performance Liquid Chromatography (HPLC)
Although the subject of chromatography is covered comprehensively in the several volumes of the Chrom-Ed Chromatography Series this page presents some of the system requirements for modern High Performance Liquid Chromatography (HPLC)
HPLC is liquid chromatography which has been optimized to provide rapid high resolution separations. It evolved over nearly a century from the early work of Tswett in the late 1900s to the highly sophisticated reliable and fast liquid chromatography (LC) techniques in common use today. Early LC used gravity fed open tubular columns with particles 100s of microns in size; the human eye was used for a detector and separations often took hours (days?) to develop. Today's HPLC requires very special apparatus which includes the following.
- Extremely precise gradient mixers (optional).
- HPLC high pressure pumps with very constant flow.
- Unique high accuracy, low dispersion, HPLC sample valves.
- Very high efficiency HPLC columns with inert packing materials.
- High sensitivity low dispersion HPLC detectors.
- High speed data acquisition systems.
- Low dispersion connecting tubes for valve to column and column to detector.
HPLC Gradient Mixers
HPLC gradient mixers must provide a very precise control of solvent composition to maintain a reproducible gradient profile. This can be complicated in HPLC by the small elution volumes required by many systems. It is much more difficult to produce a constant gradient when mixing small volumes then when mixing large volumes. For low pressure systems this requires great precision in the operation of the miniature mixing valves used and low dispersion flows throughout the mixer. For multi-pump high pressure systems it requires a very precise control of the flow rate while making very small changes of the flow rate.
Because of the small particles used in modern HPLC, modern LC pumps need to operate reliably and precisely at pressures of 10,000 p.s.i. or at least 6,000 p.s.i. To operate at these pressures and remain sensibly inert to the wide variety of solvents used HPLC pumps usually have sapphire pistons, stainless steel cylinders and return valves fitted with sapphire balls and stainless steel seats. For analytical proposes HPLC pumps should have flow rates that range from 0 to 10 ml/min., but for preparative HPLC, flow rates in excess of 100 ml/min may be required. It is extremely difficult to provide a very constant flow rate at very low flow flow rates. If .1% is considered acceptable then for 100uls/min a flow variation of less then .1ul/min is required. This level of constancy is required because most HPLC detectors are flow sensitive and errors in quantization will result from changes in flow rate.
For more general information on HPLC pumps see The LC Pump
HPLC Sample Valves
Since sample valves come between the pump and the column it follows that HPLC sample valves must also tolerate pressures up to 10,000 p.s.i. For analytical HPLC, the sample volume should be selectable from sub- micro liter to a few micro liters, whereas in preparative HPLC the sample volume may be even greater than 10 ml. To maintain system efficiency the sample valve must be designed to have very low dispersion characteristics, this is true not only for flow dispersion but also for the less obvious problems of dispersion caused by sample adsorption/desorption on valve surfaces and diffusion of sample into and out of the mating surfaces between valve moving parts. It goes without saying that the valves must deliver a very constant sample size but this is usually attained by the use of a constant size sample loop.
HPLC columns are packed with very fine particles (usually a few microns in diameter). The very fine particles are required to attain the low dispersion that give the high plate counts expected of modern HPLC. Plate counts in excess of 25,000 plates per column are possible with modern columns, however, these very high efficiencies are very rarely found with real samples because of the dispersion associated with injection valves, detectors, data acquisition systems and the dispersion due to the higher molecular weight of real samples as opposed to the common test samples. Packing these small particles into the column is a difficult technical problem but even with good packing a great amount of care must be given to the column end fittings and the inlet and outlet connection to keep dispersion to a minimum. Some state of the art systems are now 'chip' based and may use no particles at all. Some limited use has been made of HPLC for preparative purposes using half inch to one inch diameter columns. LC columns, in general, achieve their separation by exploiting the different intermolecular forces between the solute and the stationary pahse and those between the solute and the mobile phase. The column will retain those substances that interact more strongly with the stationary pahse than those that interact more strongly with the mobile phase. The basic intermolecular forces that are exploited in the HPLC are the same as those discussed in The Mechanism of Chromatographic Retention and The Thermodynamics of Chromatography of the Chrom-Ed series. The main consideration with HPLC is the much wider variety of solvents and packing materials that can be utilized as because of the much lower quantities of both which are required. In particular very expensive optically pure compounds can be used to make Chiral HPLC stationary phases and may even be used as (disposable) HPLC solvents.
LC detectors have been extensively discussed in Liquid Chromatography Detectors and HPLC detectors use the same detection principals with extra care being given to the small solute elution volumes that result from the combination of high column efficiencies with small volumes. In order to give an accurate chromatographic profile the detector sampling (cell) volume must be a small fraction of the solute elution volume. If the detector volume were larger then the elution volume then you would have peaks that appeared with flat tops as the whole peak would be resident in the detector at the same time. This means that as column volumes decrease and system efficiencies increase the volume of the detector cell volume must also decrease. This is of course at odds for the requirement for detector to maintain high sensitivity as this is usually dependant on having a larger cell volume. Again, this requires the very careful design of modern detectors.
HPLC Data Acquisition
Data acquisition was discussed in Liquid Chromatography Detectors and the only extra consideration required for HPLC is the higher sampling rate needed for the rapidly eluting narrow peaks of the HPLC chromatogram. Although the theoretical number of samples needed for good quantization are actually quite small, for real systems a hundred samples or more per peak is recommended; thus, for a 4 sec wide peak, a rate of 25 samples per second may be required. The same data analysis and reporting software can be used as in ordinary LC.
Major HPLC Systems Suppliers
HPLC is probably the most universal type of analytical procedure; its application areas include, quality control, process control, forensic analysis, environmental monitoring and clinical testing. In addition HPLC also ranks as one of the most sensitive analytical procedures and is unique in that it easily copes with multi-component mixtures. It has achieved this position as a result of the constant evolution of the equipment used in LC to provide higher and higher efficiencies at faster and faster analysis times with a constant incorporation of new highly selective column packings.