Modern HPLC essentially comprises of the following main components namely :
(i) Solvent reservoir and degassing system,
(ii) Pressure, flow and temperature,
(iii) Pumps and sample injection system,
(vi) Strip-chart recorder, and
(vii) Data handling device and microprocessor control.
All these vital components will be discussed with adequate details, wherever necessary, in the various sections that follow :
The Figure 30.1, illustrates the flow diagram of a high performance liquid chromatograph, wherein all the vital components have been duly represented.
The mobile phase, that may be either a single liquid or a mixture of two or more liquids, is pumped at high pressure into a temperature controlled oven, where it first, gains its entry into an equilibration coil to bring it to the operating temperature, and secondly, through a ‘guarded column’ specially designed and strategically positioned to protect the analytical column from impurities and ultimately extend its lifetime.
Mobile phase consisting of mixture of organic solvents or an aqueous-orgainc mixture or a buffer solution may be employed depending upon the chromatograohic method vis-a-vis the detector to be used.
There are, in fact, several factors that are solely responsible for the ‘pressure’ developed in a column, namely :
(a) the length of the column,
(b) particle size of the stationary phase,
(c) viscosity of the mobile-phase, and
(d) flow-rate of the mobile-phase.
The pressures mentioned above correspond to mobile-phase flow rates of approximately 1-5 cm3 min–1 through the column.
Flow : The flow can be measured periodically at the column outlet by collecting the liquid for a known period, and thereafter, either measuring the volume or weighing it physically.
Temperature : In reality, the maintenance of strict ‘temperature control’ plays a vital role in measuring the retention-data correctly and precisely. It makes use of the refractometer detectors specifically. In HPLC, difficult separations may be achieved by increasing the temperature carefully, but this must be done initially on a hit and trial basis.
Pumps : The two major functions of the pump in a modern HPLC are, namely :
(i) To pass the mobile-phase through the column at a high pressure, and
(ii) At a constant a controlled flow rate.
HPLC makes use of two types of pumps. They are :
(a) Constant Pressure Pump : A constant-pressure pump acts by applying a constant pressure to the mobile-phase. The flow rate through the column is determined by the flow resistance of the column.
(b) Constant Flow Pump : A constant-flow pump affords and maintains a given flow of liquid. The pressure developed entirely depends upon the flow resistance.
Importantly, in a constant-pressure pump the flow rate will change if the flow resistance changes. Whereas in the constant flow pumps the changes in flow resistance are compensated duly by a change of pressure. Therefore, it is always advisable to use constant flow pump in HPLC determinations.
Salient features of HPLC pump are as follows :
(i) Interior of the pump must not be corroded by any solvent to be used in the system,
(ii) Variable-flow-rate device must be available to monitor flow rate,
(iii) Solvent flow must be non-pulsing,
(iv) Changing from one mobile-phase to another must be convenient, and
(v) It should be easy to dismantle and repair.
The pump is a very delicate and sensitive part of HPLC unit ; therefore, all buffer solutions should be removed carefully after use either by pumping water (HPLC-grade) or an appropriate solvent (HPLC-grade) for several minutes.
Reciprocating Pump : Figure 30.2 represents the schematic diagram of a typical reciprocating pump along with its various essential components. The piston is moved in and out of a solvent chamber by an eccen-tric cam or gear. The forward-stroke closes the inlet-check value while the outlet valve opens and the respective mobile phase is duly pumped into the column. Consequently, the return-stroke-closes the outlet valve and it refills the chamber.
Advantages : It has the following advantages, namely :
(i) It has an unlimited capacity,
(ii) The internal-volume can be made very small from 10-100 μ l,
(iii) The flow-rate can be monitored either by changing the length of the piston or by varying the speed of the motor, and
(iv) It has an easy access to the valves and seals.
The use of twin-head reciprocating pump (i.e., having the two heads operated 180° out of phase) func-tions in such a manner that while one head is pumping, the other is refilling as could be seen in Figure 30.3.
In Figure 30.3, the flow rate of a twin-head reciprocal pump has been plotted against time. The stage-A depicts the drive while the refill zone is vacant ; while the stage-B evidently shows the two-heads functioning simultaneously thereby the drive and the refill both zones could be visualized.
Sample Injection System : There are in all three different modes of sample injection system that are used in HPLC, namely :
(a) Septum Injectors : They usually permit the introduction of the sample by a high pressure syringe through a self-sealing elastometer septum. The major drawback associated with this type of injec-tors is ‘leaching effect’ of the mobile-phase just in contact with the septum, thereby resulting in the formation of ‘ghost peaks’ or ‘pseudo peaks’. In short, in HPLC the mode of syringe injection brings about more problems than in GC.
(b) Stop-flow Septumless Injection : Here, most of the problems associated with septum-injectors have been duly eliminated. The flow of the mobile-phase through the column is stopped for a while, and when the column reaches an ambient pressure the top of the column is opened and the sample introduced at the top of the packing.
The first two methods are relatively very cheap.
(c) Microvolume Sampling Valves : Highly sophisticated modern HPLC frequently make use of microvolume sampling valves for injection which not only give fairly good precision, but also are adaptable for automatic injection. These valves enable samples to be introduced reproducibly into pressurized columns without causing the least interruption of the mobile-phase flow.
Figure 30.4, displays the operation of a sample loop in two different modes i.e., (a) sampling mode and (b) injection mode. Here, the sample is loaded at atmospheric pressure into an external loop in the microvolume-sampling valve, and subsequently injected into the mobile-phase by a suitable rotation to the valve. However, the volume of sample introduced usually ranges between 2 µl to over 100 µl ; but can be varied either by altering the volume of the sample loop or by employing specific variable-volume sample valves.
Therefore, it is always preferred for most quantitative work by virtue of its very high degree of precision and accuracy.
(a) Dimensions and Fillings : Following are the various dimensions of HPLC columns :
Material: Stainless-steel (highly polished surface)
External Diameter: 6.35 mm (or ≡ 0.25 inch),
Internal Diameter: 4-5 mm (usual : 4.6 mm), and
Length: 10-3 cm (usual : 25 cm).
(b) Fittings : Each end of the column is adequately fitted with a stainless-steel gauze or frit with a mesh of 2 μ m or less so as to retain the packing material (usually having a particle diameter 10, 5 and 3 μ m).
A stainless-steel-reducing union for a column of ID 4.6 mm type makes use of a 1/4-1/6 inch union with a short length of 0.25 mm (or 0.01 inch) ID ptfe tube so as to connect the column to the detector.
In actual practice, three conventional reducing unions available are employed, namely :
(i) Large Dead Volume (LDV)) Union : Loss of efficiency,
(ii) Zero Dead Volume (ZDV) Union : Loss of efficiency, and
(iii) Low Dead Volume (LDV) Union : Most efficient, most expensive, and dead-volume 0.1 μ l.
Figure 30.5 depicts the diagram of a typical LDV-Union having a SS-frit of 2 μ m and a ptfe tubing of 1/6 inch.
(i) Performance : Inside a column the concentration of a band of solute decreases as it moves through the system. The column performance or the efficiency of a column entirely depends on the amount of spreading that takes place. The measurement is represented in Figure 30.6, below :
The efficiency or performance of a column may be measured by the following expression :
N = 16(VR/WB)2 ...(a)
H = L/N ...(b)
VR = Retention volume of a solute,
WR = Volume occupied by a solute (or ‘Peak-Width’). Evidently, for a more efficient column, WB will be smaller at a given value of VR,
H= Plate number of the column (dimensionless), H = Plate height of the column (mm × μ m), and L = Length of the column (cm).
Based on Eq. (b) one may clearly observe that for a more efficient column ‘N’ gets larger and corre-spondingly ‘H’ gets smaller.
(iii) Types of Packing : Modern HPLC makes use of packing which essentially consist of small and rigid particles with a very narrow particle size distribution. Broadly speaking three types of packing are invariably used in HPLC column, namely :
(a) Styrene-divinylbenzene copolymers based porous polymeric beads have been employed exclusively for size-exclusion and ion-exchange chromatography, but now mostly been replaced by silica-based packings that proved to be more efficient and mechanically stable.
(b) Porous-layer beads with a diameter ranging between 30-35 μ m comprising of a thin shell (1-3 μ m) of silicon or modified silica, on an inert spherical core material, such as : glass beads are still being employed for certain ion-exchange procedures ; but of late their usage as such in HPLC have been superseded by 100% porous microparticulate packings, and
(c) Porous-silica particles (100%) with a diameter less than 1 μ m and narrow-particle size range, nowadays, form the basis of most abundantly available important column packings used in analytical HPLC. In comparison to the porous-layer beads, as detailed in (b) above, the porous-silica particles yield significant improvements not only in column efficiency but also in sample capacity and speed of analysis.
The main function of the detector in HPLC is to monitor the mobile-phase coming out of the column, which in turn emits electrical signals that are directly proportional to the characteristics either of the solute or the mobile-phase.
The various detectors often used in HPLC may be categorized into three major heads, namely :
(i) Bulk-property detectors : They specifically measure the difference in some physical property of the solute present in the mobile-phase in comparison to the individual mobile-phase, for instance :
(a) Refractive-index detectors, and
(b) Conductivity detectors.
(ii) Solute-property detectors. They critically respond to a particular physical or chemical characteristic of the solute (in question), which should be ideally and absolutely independent of the mobile-phase being used. But complete independence of the mobile-phase is hardly to be seen, however, signal discrimination is good enough to enable distinctly measurable experimental procedures with solvent changes, such as : gradient-elution.
The solute-property detectors include :
(a) UV-detectors, and
(a) Fluorescence Detectors.
(iii) Multipurpose detectors : Besides, providing a high degree of sensitivity* together with a broad-linear-response-attainable range, invariably a particular situation critically demands detectors of more selective nature in the domain of ‘analytical chemistry’ vis-a-vis ‘Pharmaceutical Analysis’ that could be accomplished by using ‘multipurpose detectors’, such as : “Perkin-Elmer ‘3D’ Sys-tem” that combines UV absorption, fluorescence and conductometric detection.
(iv) Electrochemical detectors : ‘Electrochemical detector’ in HPLC usually refers to either amperometric or coulometric detectors, that specifically measure the current associated with the reduction or oxidation of solutes. As only a narrow spectrum of compounds undergo electrochemical oxidation, such detectors are quite selective ; and this selectivity may be further enhanced by moni-toring the potential applied to the detector so as to differentiate between various electroactive spe-cies. Naturally, electrochemical detection essentially makes use of conducting mobile phases, for instance : inorganic salts or mixtures of water with water-miscible organic solvents.
The five important types of detectors shall be discussed along with their simple diagrammatic sketches, in the sections that follow :
Principle : An UV-detector is based on the principle of absorption of UV visible light from the effluent emerging out of the column and passed though a photocell placed in the radiation beam.
Figure 30.7 represents the schematic diagram of a double-beam UV detector used in HPLC system. Initially, dual-wavelength instruments having 254 and/or 280 nm were introduced which is presently being replaced by more sophisticated and up-dated variable wavelength detectors spread over wide range between 210-800 nm capable of performing more selective detection possible.
Diode Array Detector (or Multichannel Detector) is also a UV detector wherein a polychromatic light is made to pass through the flow cell. A strategically placed grating diffracts the outcoming radiation and subsequently meets an array of photodiodes whereby each photodiode receives a different narrow wavelength band. Here, a microprocessor scans the array of diodes several times in one second and the resulting spectrum is visualized on the screen of a VDU or subsequently stored in the instrument for a printout as and when required. Another extremely important and useful characteristic feature of a diode-array detector is that it may be ‘programmed’ so as to affect changes in the detection wavelength at particular points in the chromatogram. This versatile criterion is used to ‘clean up’ a chromatogram i.e., to discard all interfering peaks caused due to components irrelevantly present in the sample.
Advantages : Various advantages are, namely :
(a) A very selective detector which will detect only such solutes that specifically absorb UV/visible radiation e.g., alkenes, aromatics and compounds having multiple bonds between C, O, N and S.
(b) The mobile-phase* employed ideally must not absorb any radiation.
A plethora of compounds (solutes) present in the mobile-phase on being passed as column effluent through a cell irradiated with Xenon or Deuterium source first absorb UV radiation and subsequently emit-radiation of a longer wavelength in two different manners, namely :
(a) Instantly-termed as ‘Fluorescence’, and
(b) After a time-gap-known as ‘Phosphorescence’.
Fluorescent compounds : A relatively small proportion of inorganic and organic compounds exhibit natural fluorescence, whereas a larger number of pharmaceutical substances and environmental contaminants [e.g., polycyclic aromatic hydrocarbons (PAH)] having a conjugated-cyclic system are fluorescent. Such com-pounds having absorbed energy being re-emitted from 0.1-1.0 can be detected by a fluorescence detector. However, non-fluorescent compounds can be converted to fluorescent derivatives by treatment with appropri-ate solvents.
Figure 30.8, illustrates the diagram of a fluorescence detector :
Radiation from a Xenon-radiation or a Deuterium-source is focussed on the flow cell through a filter. The fluorescent radiation emitted by the sample is usually measured at 90° to the incident beam. The second filter picks up a suitable wavelength and avoids all scattered light to reach ultimately the photomultiplier detector.
It is also known as ‘RI-Detector’ and ‘Refractmeter’. Figure 30.9, represents the block-diagram of a refractive-index detector.
Light from the source(s) is focused into the cell, that consists of sample and reference sample ; and the two chambers are separated by a diagonal sheet of glass. After passing through the cell, the light is diverted by a beam-splitter (B) to two photocells (P1 and P2 respectively. A change in the observed refractive index (RI) of the sample stream causes a difference in their relative output, which is adequately amplified and recorded duly.
The RI of a few commonly used mobile-phase is stated below :
Any solute can be detected as long as there exists a measurable difference in refractive index between the solute and the mobile-phase.
A multipurpose detector essentially comprises of three detectors combined and housed together in a single unit. A typical example of such a detector is the one developed by Perkin-Elmer known as “Perkin-Elmer ‘3D’ System” which is depicted in Figure 30.10.
The functions of the three different detectors used in Figure 30.10 are enumerated as under :
(i) Fluorescence Function : It can monitor emission above 280 nm, based on excitation at 254 nm,
(ii) UV-Function : It is fixed wavelength 254 nm detector, and
(ii) Conductance-Function : The metal inlet and outlet tubes serve as electrodes to measure the con-ductance of the ions.
In actual practice, however, it is rather difficult to utilize the functions of electrochemical reduction as a means of detection of HPLC by virtue of the fact that the serious interference (i.e., large background current) generated by reduction of oxygen in the mobile phase. As complete removal of oxygen is almost difficult, therefore, electrochemical detection is normally based upon the oxidation of the solute.
Examples : The various compounds that may be detected conveniently are, namely : aromatic amines, phenols, ketones, and aldehydes and heterocyclic nitrogen compounds.
In short, the amperometric detector is presently considered to be the best electrochemical detector having the following distinct advantages, such as :
(i) very small internal cell-volume,
(ii) high degree of sensitivity,
(iii) more limited range of applications, and
(iv) excellent for trace analyses as UV-detector lacks adequate sensitivity.
Table 30.2, provides a comprehensive comparison of various typical detector characteristics invariably used in HPLC, such as : response, concentration expressed in g ml–1 and the linear range. However, the linear range usually refers to the range over which the response is essentially linear. It is mostly expressed as the factor by which the lowest factor (i.e., Cn) should be multiplied in order to obtain the highest concentration.
The signal emerging from the detector of a HPLC is recorded continuously as function of time most commonly with the help of a potentiometric recorder. Invariably, a recorder of 1 to 10 mV full-scale deflec tion over a stretch of approximately ten inches and having a response-time of one second or even less is regarded as most appropriate. Strip-chart recorder with variable chart speeds ranging between 5 to 5 mm min–1 are usually preferred.
The input signal of a potentiometric-recorder is balanced continuously with the help of a feedback signal arrangement (device) using a servomechanism. A pen attached to this device moves proportionately, with preadjusted attenuation, along the width of the chart-paper thereby recording the signal accurately, while the chart-paper moves at a fixed speed along the length.
It is pertinent to mention here that before commencing the operation of a recorder, its zero point must be adjusted with the input zero, otherwise the baseline will also shift with slight changes in the attenuation of the signal.
Besides, it is also equally important to adjust properly the amplifier gain so as to eliminate completely the dead-band and the oscillations. A recorder having inadequate shielding from the AC circuits may display shifting of its zero point.
Modern HPLC is adequately provided with complete data handling devices. Thousands of samples routinely analysed in Quality Assurance Laboratories in Pharmaceutical Industries/Bulk Drug Industries etc. are duly processed and the data stored in the computerised data-handling devices. Each stored data may be retrieved from the memory of the computerised device with the flick of a finger, as and when needed, in the form of print-out.
Microprocessor based analytical equipments is no longer an uncommon phenomenon towards the mod-ernization, automation, and above all the ease of function and handling of sophisticated devices, for instance : a microprocessor scans the array of diodes many times a second in a ‘diode array detector’ ; a microproces-sor does the temperature programming of a constant temperature chamber of HPLC unit.