Syringe pump, HPLC

The instrument was built by modifying the design of a TREF 300 unit (Polymer Char, Spain) which incorporates an oven used for sample preparation and a high precision TREF column oven. Other components are syringe pump, HPLC pump, high temperature isothermal oven (SEC oven) in which the injection valve, a multiposition selection valve and the SEC column set are placed. A dual band 1R4 infrared detector (Polymer Char, Spain) is used as the cOTicentration detector. 

The types of pumps used for HPLC can be divided into two categories constant-pressure pumps (e.g. the inexpensive gas-displacement pump) and the constant-volume type (e.g. the reciprocating and syringe pumps). The most commonly used pumps in HPLC are the single- or multi-head reciprocating type. The former delivers the flow as a series of pulses which must be damped... 

Implementation of SFC has initially been hampered by instrumental problems, such as back-pressure regulation, need for syringe pumps, consistent flow-rates, pressure and density gradient control, modifier gradient elution, small volume injection (nL), poor reproducibility of injection, and miniaturised detection. These difficulties, which limited sensitivity, precision or reproducibility in industrial applications, were eventually overcome. Because instrumentation for SFC is quite complex and expensive, the technique is still not widely accepted. At the present time few SFC instrument manufacturers are active. Berger and Wilson [239] have described packed SFC instrumentation equipped with FID, UV/VIS and NPD, which can also be employed for open-tubular SFC in a pressure-control mode. Column technology has been largely borrowed from GC (for the open-tubular format) or from HPLC (for the packed format). Open-tubular coated capillaries (50-100 irn i.d.), packed capillaries (100-500 p,m i.d.), and packed columns (1 -4.6 mm i.d.) have been used for SFC (Table 4.27). 

TMSCN (1.8 equiv) was added over 10 h via syringe pump unless otherwise mentioned. 4 Isolated yield.c Determined by HPLC analysis. Configuration assigned by comparison to literature values of optical rotation. d 20 mol % of 123 and 80 mol % of the addtive were used. 1.2 equiv of TMSCN was used. e TMSCN (1.2 equiv) was added dropwise over 1 min. f 18 mol % of 123 and 72 mol % of the additive waer used. The absolute configuration was not determined. 

Syringe pumps driven by screw mechanisms were popular in the 1960s because of their inherent precision and pulseless flow characteristics. Their disadvantages are higher manufacturing costs and the problems associated with syringe refill cycles. Syringe pumps are currently used in specialized systems for microbore and capillary HPLC. 

In addition to the commercially available systems, several authors have described laboratory-built systems using commercially available components from companies such as Upchurch Scientific (Oak Harbor, WA). One of the first reported laboratory-built micro-bore HPLC systems was described by Simpson and Brown, which was a simple adaptation of a standard HPLC system to accept micro-bore columns built from guard columns. A complete system has been described based on dual microdialysis syringe pumps (CMA Microdialysis, Chelmsford, M A) or dual syringe pumps (Harvard Apparatus, Inc., Holliston, MA), a microinjection port, and a micro-column the latter components being obtained from Upchurch scientific (Figure 3.5). This system was coupled with a laser-induced fluorescence (LIF) detector and used to measure neuropeptides in sub-microliter samples. A further modification of this system was built to perform immunoaffinity isolations of biomedically important analytes from clinical samples. ... 

FIGURE 3.5 (A) A laboratory-built J,HPLC system with dual syringe pumps, an electronic injector port,... 

SFC chromatographs represent hybrids between GC and HPLC instruments (Fig. 6.4). In order to deliver the supercritical fluid, syringe pumps or reciprocal pumps are used and maintained above the critical temperature using a cryostat regulated at around 0 "C. In instances where an organic modifier is used, a tandem pump is employed which has two chambers, one for the critical fluid and one for the modifier. The liquid then passes through a coil maintained above the critical temperature so that it is converted into a supercritical fluid. Stainless steel packed columns like those used in HPLC (1 to 4 mm in diameter) or fused silica capillary columns like those used in capillary GC (2 to 20 m in length, internal diameters as low as 50 pm and stationary phase film thickness of at least 1 pm) are used in SFC. 

The SFE process was carried out in a JASCO system. For each run, which lasted for an hour, 0.5g of the as-synthesised HMS was being loaded into the extraction cell housed in an oven. The system uses a HPLC and a syringe pump for pumping liquid CO2 and the modifier (methanol) respectively so as to build up the system pressure. The desired system pressure was set and controlled by a back pressure regulator while the system temperature was set and controlled by the temperature controller attached to the oven. The extracted amine surfactant is collected in a vial placed at the outlet of the back pressure regulator. 

A reversed-phase HPLC column (typically Cl 8 or C30) is required for HPLC separations. Because the flow rate into the continuous-flow FAB-MS or LSIMS source must be <10 pl/min, either a capillary column must be used or else the flow must be split postcolumn. For narrow-bore HPLC columns operated at 200 pl/min, the split ratio would be 30 1. Isocratic or gradient separations may be used. A syringe pump is usually necessary for capillary columns, but standard HPLC pumps are sufficient for applications using narrow-bore columns. 

A reversed-phase HPLC Cl8 or C30 narrow-bore column is typically used for LC/MS with APCI. Details about chromatography columns used for carotenoids are contained in unit F2.3. For most APCI systems, the optimum flow rate into a mass spectrometer or tandem mass spectrometer equipped with APCI, as controlled by a syringe pump or HPLC pump, is usually between 100 and 300 pl/min, which is ideal for narrow-bore HPLC columns. Larger diameter columns should be used with a flow splitter postcolumn to reduce the solvent flow into the mass spectrometer. For example, if a 4.6 mm i.d. column was used at a flow rate of 1.0 ml/min, then the flow must be split postcolumn 5 1 so that only 200 pl/min enters the mass spectrometer. 

Syringe pump or HPLC pump capable of flow rates of 1 to 10 pl/min Reversed-phase (typically Cl8) column (since flow rate into continuous-flow FAB source must be <10 pl/min, capillary column must be used or flow must be split post column)... 

Reactant feeds are generated by vaporizing liquid flows from HPLC pumps with manometric pulse dampeners or high-pressure syringe pumps and mixing this vapor with gas components metered through mass flow controllers. This reaction feed is then divided between the 48 channels equally by using flow restrictors such as silica capillaries or micromachined channels (Fig. 3.12). The capillaries feed into the inlet stand-offs of the reactor modules. 

Two factors are driving the market for precise, very-low-flow HPLC pumping systems extremely limited sample sizes in biotechnology and the electrospray and nanospray interfaces that are concentration and flow-rate dependent. It is very difficult to get precise flow and gradient formation from pumps that have a 5- to 10-/iL plunger displacement, even using 3200-step stepper motor drives. This has forced manufacturers to resurrect a very old concept from the earliest days of HPLC, the syringe pump. 

Nanoflow HPLC—HPLC system with accurately controlled reciprocating and syringe pumps designed to use capillary and small diameter, high-resolution columns as front ends for electrospray and nanospray mass spectrometer interfaces. 

Syringe Pump—A pulseless pump made up of a motor-driven piston or plunger in a solvent-filled cylinder. Useful only when small solvent volumes are to be pumped often used in micro-flow or nano-flow HPLC systems. 

Figure 6.8 Diagram of instrumental configuration of the LC/MS system used for characterization of crude fermentation extracts. The system consists of the following components (1) HPLC (2) loop injector (3) guard column (4) 5pm C18 HPLC column (4.6mm x 25cm) (5) zero dead volume tee (6) UV detector (7) fraction collector (8) triple quadrupole mass spectrometer equipped with ESI interface (9) ESI power supply and gas manifold and (10) syringe pump. (Reprinted with permission from Ackermann et al., 1996a. Copyright 1996 Elsevier.)...

Fig. 1 Using a T-piece between HPLC and MS, a syringe pump can be utilized to infuse a constant flow of analytes at therapeutic concentrations whilst drug spiked whole blood samples are delivered via SPE-HPLC. Ion traces of the analytes (here everolimus at 6 ng/ml) are recorded. In this particular case, no ion yield attenuation due to the spiked drug can be observed, although strong effects can be seen in the solvent front elution zone shortly prior to the analyte elution time window...

FIGURE 12 Three platforms for automated solid-phase oligosaccharide synthesis, (a) Pressure-driven system, (b) Syringe pump-driven system, (c) HPLC pump-driven system. 

FIGURE I (A) An ESI source attached to a single quadrupole analyzer. The Instrument can be interfaced to an HPLC, CE or a syringe pump. The ionization is done at atmospheric pressure and the mass analysis in vacuum. This configuration is used for molecular weight determination. (B) An ESI source attached to a triple quadrupole analyzer. In a typical MS - MS experiment the precursor ion (Mp) is selected by the first analyzer (Ql), reacts with an inert gas in Q2 to yield the product ions (MI, M2, M3), and the product ions are analyzed by scanning Q3. This yields the product (fragment) ion spectrum. 

Peak identification and interpretation of mass spectra Mobile phases for proanthocyanidins contain 2% (v/v) of acetic acid, and it suppresses electrospray ionization at negative mode. This can be overcome by adding ammonia acetate as an ionization enhancer. The ammonia acetate (lOmM in methanol, 0.1 mL/min flow rate) can be added into the flow via a three-way micro-splitter ( P-445, Upchurch Scientific, WA) just before the mass spectrometry. It can be delivered by a separate HPLC pump or by a syringe pump. 

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