86 BASIC CONCEPTS AND THE CONTROL OF SEPARATION 85. J T.Lin,L.R.Snyder,andT.A.McKeon,J. Chromatogr. A, 808 (1998) 43. 86. C. T. Mant and R. S. Hodges, in HPLC of Proteins, Peptides and Polynucleotides,M. T. W. Hearn, ed., VCH, New York, 1991, p. 277. 87. K. Yanagida, H. Ogawa, K. Omichi, and S. Hase, J. Chromatogr. A, 800 (1998) 187. 88. T. Baczek, R. Kaliszan, H. A. Claessens, and M. A. van Straten, LCGC Europe,13 (2001) 304. 89. V. Spicer, A. Yamchuk, J. Cortens, S. Sousa, W. Ens, K. G. Standing, J. Q. Wilkens, and O. V. Korkhin, Anal. Chem., 79 (2007) 8762. 90. P. C. Sadek, P. W. Carr, R. M. Doherty, M. J. Kamlet, R. W. Taft, and M. H. Abraham, Anal. Chem., 57 (1985) 2971. 91. C. F. Poole and S. K. Poole, J. Chromatogr. A, 965 (2002) 263. CHAPTER THREE EQUIPMENT 3.1 INTRODUCTION, 88 3.2 RESERVOIRS AND SOLVENT FILTRATION, 89 3.2.1 Reservoir Design and Use, 90 3.2.2 Mobile-Phase Filtration, 91 3.3 MOBILE-PHASE DEGASSING, 92 3.3.1 Degassing Requirements, 92 3.3.2 Helium Sparging, 94 3.3.3 Vacuum and In-line Degassing, 95 3.4 TUBING AND FITTINGS, 96 3.4.1 Tubing, 96 3.4.2 Fittings, 99 3.5 PUMPING SYSTEMS, 104 3.5.1 Reciprocating-Piston Pumps, 104 3.5.2 On-line Mixing, 109 3.5.3 Gradient Systems, 112 3.5.4 Special Applications, 112 3.6 AUTOSAMPLERS, 113 3.6.1 Six-Port Injection Valves, 114 3.6.2 Autosampler Designs, 116 3.6.3 Sample-Size Effects, 119 3.6.4 Other Valve Applications, 122 3.7 COLUMN OVENS, 125 3.7.1 Temperature-Control Requirements, 125 3.7.2 Oven Designs, 126 3.8 DATA SYSTEMS, 127 3.8.1 Experimental Aids, 127 3.8.2 System Control, 129 3.8.3 Data Collection, 129 3.8.4 Data Processing, 130 3.8.5 Report Generation, 130 3.8.6 Regulatory Functions, 130 3.9 EXTRA-COLUMN EFFECTS, 131 Introduction to Modern Liquid Chromatography, Third Edition, by Lloyd R. Snyder, Joseph J. Kirkland, and John W. Dolan Copyright © 2010 John Wiley & Sons, Inc. 87 88 EQUIPMENT 3.10 MAINTENANCE, 131 3.10.1 System-Performance Tests, 131 3.10.2 Preventive Maintenance, 138 3.10.3 Repairs, 143 3.1 INTRODUCTION Equipment design for modern HPLC is in a mature state. With certain exceptions (e.g., high-pressure applications, Section 3.5.4.3), major changes in equipment design and features are not often encountered. While small changes from one model to its replacement continue to improve the reliability of HPLC equipment, the rapid obsolescence of HPLC equipment that was once a concern is no longer an issue for most applications. Analysts beginning their use of HPLC often ask which system or manufacturer is ‘‘best.’’ Today there is less distinction between HPLC systems than in the past, and it can be safely said that there are no ‘‘bad’’ HPLC systems currently on the market. This means that a features-and-benefits approach to equipment selection often gives way to choices based on local service and support provided by the equipment vendor. Users in the past often would select specific equipment modules from different vendors and, in a mix-and-match approach, would design their own ‘‘ideal’’ HPLC system. Today this is not common, partly because of the equivalent performance of components between manufacturers, and partly because of the interdependence of the various modules. Usually components chosen from a single manufacturer will work together better than will modules from several manufacturers combined into a single system. Thus the pump, autosampler, and column oven usually are obtained as a unit or as compatible components from a single manufacturer. The detector may be obtained from a second manufacturer, especially for specialty detectors, such as MS/MS (Section 4.10). Because the major data-system manufacturers often include the ability to control equipment from other vendors, the data system may be chosen from another manufacturer than the pumping compo- nents. However, when maintenance, training, repair, and equipment compatibility are considered, most laboratories purchase as many components of the HPLC system as possible from a single vendor and stay with a single manufacturer if multiple HPLC systems are operated in a single facility. An alternative practice is used in some large laboratories, especially those that transfer methods to other sites (Section 12.7). In such cases equipment is selected from several manufacturers in order to allow comparison of method performance on different instruments. This approach helps highlight potential equipment-dependent method-transfer problems that can be addressed prior to transfer of the method to a second laboratory. 3.2 RESERVOIRS AND SOLVENT FILTRATION 89 Figure 3.1 HPLC system diagram. The essential components of an HPLC system are shown in Figure 3.1. Mobile phase is drawn from a reservoir into a pump, which controls the flow rate and generates sufficient pressure to drive the mobile phase through the column. An injector or autosampler is used to place the sample on the column without stopping the pump flow. The separation takes place in the column, which generally resides inside a column oven. The detector responds to changes in analyte concentration during the run. A data system monitors the detector output and provides data processing for both graphic and tabular output of data. A system controller (often combined with the data system) directs the functions of the various modules. The HPLC system may comprise a group of individual components (often referred to as a ‘‘modular’’ system), or the components may be combined within a single cabinet as an ‘‘integrated’’ system. Because of the precious nature of laboratory bench space, modular systems usually are designed to enable stacking of components for a small footprint, similar to that of an integrated system. In addition to systems designed for analytical applications, HPLC systems may be specially designed for low-flow (micro), high-flow (preparative), or high-pressure applications (Sections 3.5.4 and 15.2). The majority of analytical methods rely on UV detection, but many other detectors are available for specialized applications (Chapter 4). In this chapter the various components of the HPLC system are discussed, with the exceptions of the detector (Chapter 4) and application of the data system (Chapter 11). Unless stated otherwise, commercially available equipment is assumed in every case. 3.2 RESERVOIRS AND SOLVENT FILTRATION Mobile-phase reservoirs (Fig. 3.2) are simple yet essential parts of the HPLC system. For isocratic applications using premixed mobile phase, only a single reservoir is 90 EQUIPMENT vent reservoir inlet-line frit (b) (a) Figure 3.2 Mobile-phase reservoir. needed. When isocratic mobile phases are blended online or for gradient applications, more than one reservoir is used. Mobile phases must be free of particulate matter, so mobile-phase filtration may be required prior to filling the reservoir. 3.2.1 Reservoir Design and Use Most reservoir containers (Fig. 3.2a) are made of glass, although some applications, such as the determination of Na + ions by ion chromatography, require a glass-free system. Laboratory glassware (e.g., Erlenmeyer flasks), heavy-walled glass bottles, or the glass bottles in which the solvents are shipped are the common reservoirs. Some equipment manufacturers supply reservoirs specifically designed for their equipment. Besides inertness to the mobile phase, cleanliness is the most important reservoir requirement. Glassware should be washed on a regular basis (e.g., weekly), using standard laboratory dishwashing techniques. A cover of some sort should be used to keep dust from entering the reservoir and to minimize evaporation of the mobile phase, but the reservoir should not be so tightly capped that a vacuum forms when mobile phase is pumped out. A threaded cap with an oversized hole (Fig. 3.2a) for the mobile-phase inlet line or a piece of aluminum foil crimped around the top of the reservoir are the most popular closure techniques and allow rapid pressure equalization when mobile phase is pumped out. The use of polymeric laboratory film products (e.g., Parafilm ® ) to cover the reservoir should be avoided, since some mobile phases may extract components that can contaminate the system. An inlet-line frit (Fig. 3.2a,b) is used at the inlet end of the tubing that connects the reservoir and the pump. The frit acts as a weight to keep the inlet tubing at the bottom of the reservoir, but its primary function is to provide backup filtration to remove particulate matter, such as dust, that might enter the reservoir. Since it is not the primary solvent filter, it should not restrict solvent flow to the pump. A frit 3.2 RESERVOIRS AND SOLVENT FILTRATION 91 porosity of ≥10 μm is recommended so that solvent can flow freely through the inlet-line frit. This can be confirmed with a siphon test. Disconnect the tube fitting at the pump inlet (high-pressure mixing systems) or solvent proportioning module (low-pressure mixing); if solvent is not flowing freely, start a siphon flowing with a pipette bulb. A good rule of thumb is that the flow through the siphon should be ≥10× the required flow rate when the solvent reservoirs are located > 50 cm above the point of measurement. For example, if flow rates of 1 to 2 mL/min are typically used, the siphon test should supply > 20 mL/min of solvent. Generally, flow rates of > 50 mL/min are observed under these conditions. If the siphon delivery is too slow, replace the frit and/or clear any blockage in the tubing. In use, the reservoir should be located higher than the pump inlet (e.g., > 50cm)soastoprovidea positive-pressure feed of solvent to the pump for more reliable pump operation. There are many designs of inlet-line frits available, and these are made of stainless-steel, ceramic, PEEK, and other materials that are inert to the mobile phase. One popular design is sketched in Figure 3.2b, in which the intake portion of the frit is on the bottom rather than the sides. This ‘‘last drop’’ design enables the use of more mobile phase in the reservoir before it must be replenished. 3.2.2 Mobile-Phase Filtration The operation of several parts of the HPLC system can be compromised if particulate matter is present. These parts include proportioning valves, check valves, tubing, and column frits. For this reason it is important to use a particulate-free mobile phase. If prefiltered (i.e., HPLC-grade) solvents are not available, the mobile-phase components should be filtered prior to adding them to the reservoir. For laboratories that work in a regulated environment, a standard operating procedure (SOP) should be written to describe when additional mobile-phase filtration is required and when it is not. Use of prefiltered solvents is the simplest way to avoid introducing particulate matter into the mobile phase. Commercial HPLC-grade solvents are filtered through submicron filters (generally 0.2 μm) prior to packaging. HPLC-grade water prepared in the laboratory (e.g., Milli-Q water purification) is passed through a final 0.2-μm filter as the last step in purification. If only HPLC-grade liquids are used in the mobile phase, it is common practice not to perform any additional filtration prior to use. However, if non–HPLC-grade reagents or any solid reagents are added to the mobile phase (e.g., phosphate buffer), it is wise to filter all mobile-phase mixtures prior to use. Mobile phases can be easily filtered with a vacuum-filter apparatus, such as that shown in Figure 3.3. A membrane filter (typically ≈0.5-μm porosity) is mounted on a support frit between the funnel and the vacuum flask. Solvent is poured into the funnel and collected under vacuum-assisted (e.g., water aspirator) filtration. Filter manufacturers provide guides to the selection of the proper filter material for each application. For example, PTFE filters are hydrophobic and work well with pure organic solvents, such as MeOH or ACN, but are too nonpolar to allow rapid filtration of water. The seal between the vacuum flask is made with a ground-glass fitting or an ‘‘inert’’ stopper (e.g., silicone), but it is best not to allow mobile phase to contact the stopper. 92 EQUIPMENT vacuum flask vacuum filter funnel support frit membrane filter inert stopper Figure 3.3 Vacuum apparatus for mobile phase filtration. 3.3 MOBILE-PHASE DEGASSING The presence of air bubbles in the mobile phase is a common problem in the operation of an HPLC system. These bubbles can lead to pump-delivery problems and spurious peaks in the detector output. Most often concern about bubbles can be eliminated by degassing the mobile phase prior to use. 3.3.1 Degassing Requirements As long as air remains dissolved in the mobile phase, bubble problems are seldom encountered. In principle, hand-mixed isocratic mobile phases should be suitable for use without degassing, but an air-saturated solution may outgas with only a minor drop in pressure, such as when the mobile phase is pulled through the solvent inlet-line filter or when it enters the relatively low-pressure region in the detector cell. For this reason, and for general HPLC operational reliability, degassing of all solvents used for reversed-phase applications is strongly recommended. Outgassing is less of a problem with normal-phase HPLC, so degassing may be considered as optional in such applications. The amount of dissolved gas that must be removed from the mobile phase will vary with the design of the HPLC pump—some pumps are very tolerant to dissolved gas, whereas others require thorough degassing for reliable operation. Bubble formation can be especially problematic in the case of mobile phases for reversed-phase chromatography (RPC), as illustrated by the data of Figure 3.4. For example, assume that pure water and pure ethanol are each saturated with oxygen, as might be the case if the solvents are exposed to air. When the solvents are blended, the mixture contains an amount of oxygen and solvent that is proportional 3.3 MOBILE-PHASE DEGASSING 93 Figure 3.4 Solubility of oxygen in ethanol. (- - -), Oxygen concentration following mixing of air-saturated water and ethanol (before release of excess oxygen); (—), saturation concentra- tion of oxygen in mixture. Adapted from [1]. to the relative concentrations of each solvent (represented by the dashed line in Fig. 3.4). However, oxygen is seen to be less soluble in a solvent mixture (solid line in Fig. 3.4), so the mixture is now supersaturated with oxygen. In such cases oxygen either bubbles out immediately or when it contacts a nucleation site, such as the rough surface of the solvent inlet-line filter. Although Figure 3.4 shows data for oxygen, water, and ethanol, the same principle holds for air (comprising primarily nitrogen and oxygen), buffered water, and other organic solvents, such as acetonitrile or methanol [1]. These data also suggest that it is not necessary to remove all of the dissolved air from solution—just enough that the amount of dissolved air in the mixtures is below the (solid) saturation curve of Figure 3.4. For most applications, degassing is important primarily to improve pump operation. However, in some cases the presence of dissolved oxygen can degrade detector performance. It has been reported [2] that UV detection (Section 4.3) as low as 185 nm is possible if the detector (and acetonitrile/water mobile phase) is purged with helium to remove oxygen from the optical path of the detector. Under these conditions the apparent detector-lamp response increased and the baseline noise was reduced. Even at higher wavelengths, dissolved oxygen in the mobile phase can elevate the detector background signal, as can be seen in Figure 3.5a. At 254 nm, the mobile phase sparged with air gave an increased baseline signal compared to the mobile phase sparged with helium, presumably because of a change in refractive index of the air-sparged mobile phase. Under the same conditions, but with fluorescence detection (Section 4.5), ≈75 % of the signal intensity for naphthalene was lost (Fig. 3.5b) when the mobile phase was sparged with air instead of helium [1]. When the electrochemical detector (Section 4.6) is operated in the reductive mode, dissolved oxygen creates an unacceptable background signal, so oxygen must be removed from the mobile phase, as by helium sparging (Section 3.3.2). Finally, it is conceivable that dissolved oxygen might react with some samples during separation. So it is important to select a degassing technique that addresses both chemical 94 EQUIPMENT Figure 3.5 Effect of helium sparging on detector response to naphthalene. (a) UV detec- tion at 254 nm, (b) fluorescence detection at 250-nm excitation and 340-nm emission. He, mobile-phase sparging with helium begins; air, sparging with air begins. Adapted from [1]. problems (e.g., detector response) and physical problems (e.g., bubbles in the pump) that may result from the presence of dissolved gas in the mobile phase. When off-line degassing is used, such as stand-alone helium sparging or vacuum degassing, the solvent will begin to re-equilibrate with air as soon as the degassing treatment is stopped. For HPLC systems that are highly susceptible to dissolved gas in the mobile phase, off-line degassing may not be sufficient. In such cases continuous helium sparging (Section 3.3.2) or on-line vacuum degassing (Section 3.3.3) are better choices. 3.3.2 Helium Sparging Helium sparging is the most effective technique for removing dissolved gas from the mobile phase [3] (with the exception of refluxing or distillation), and removes 80–90% of the dissolved air. Typically a frit is used to disperse helium (e.g., at ≈5 psi through a sparging frit) in the reservoir. Under these conditions it takes only one volume of helium to degas an equal volume of mobile phase [4]. This means that just a few minutes of a vigorous sparging stream will adequately degas the mobile phase. Helium itself has such a low solubility in HPLC solvents that a helium-sparged solution is nearly gas free. Excessive sparging of the mobile phase is undesirable, since it can change the composition of the mobile phase through evaporation of the more volatile component(s); however, vigorously sparging a RPC mobile phase for a few minutes is unlikely to cause problems Normal-phase solvents are much more volatile, so helium sparging of a (blended) mobile phase should be used cautiously—if at all. Sparging pure solvents prior to on-line mixing poses no problem, however. 3.3 MOBILE-PHASE DEGASSING 95 3.3.3 Vacuum and In-line Degassing For most HPLC systems, the application of a partial vacuum to the mobile phase will remove a sufficient amount of dissolved gas to avoid outgassing problems. Vacuum degassing for 10 to 15 minutes will remove 60–70% of the dissolved gas [3]. In its simplest form, some vacuum degassing takes place during solvent filtration, as in Figure 3.3. This can be enhanced after filtration is complete by replacing the filter funnel with an inert stopper and applying the vacuum for a few more minutes. Some users find that placement of the vacuum flask in an ultrasonic cleaning bath during this process further enhances degassing. Today in-line (or on-line) degassing is the most popular degassing technique; most HPLC equipment manufacturers include an in-line degasser as either standard or optional equipment with new systems. The operation of the in-line degasser is illustrated in Figure 3.6 for two solvents (A and B; degassers for 1–4 solvents are available), and it is based on the selective permeability of certain polymeric tubing to gas. The degasser is mounted before the pump(s) (high-pressure mix- ing, Section 3.5.2.1; or hybrid systems, Section 3.5.2.3) or proportioning valves (low-pressure mixing, Section 3.5.2.2). Solvent is passed through a piece of poly- meric tubing inside a vacuum chamber; the vacuum pulls the dissolved gas passes through the walls of the tubing; the liquid mobile phase stays inside the tubing (detail gas-permeable tubing mobile phase vacuum vacuum dissolved gas mobile phase in in out mobile phase out B A (b) (a) Figure 3.6 Diagram of a membrane degassing apparatus for two solvents, A and B. . 130 3.8.5 Report Generation, 130 3.8.6 Regulatory Functions, 130 3.9 EXTRA-COLUMN EFFECTS, 131 Introduction to Modern Liquid Chromatography, Third Edition, by Lloyd R. Snyder, Joseph J. Kirkland,. from a reservoir into a pump, which controls the flow rate and generates sufficient pressure to drive the mobile phase through the column. An injector or autosampler is used to place the sample. the pump. The frit acts as a weight to keep the inlet tubing at the bottom of the reservoir, but its primary function is to provide backup filtration to remove particulate matter, such as dust,