Solvent delivery systems, HPLC

HPLC has also been used to determine the residue levels of dinifroaniline herbicides. Pendimethalin was quantified by HPLC under the following conditions apparatus, Spectroflow 400 solvent delivery system. Model 430 gradient former, and Kratos Model 783 with UV absorbance detection at 239 nm column, Cig reversed-phase (25cmx 3.0-mm i.d.) temperature, 40 °C mobile phase, acetonitrile-water (7 3, v/v) flow rate, 1 mL min . ... 

Figure 4.31(b) from Euston and Glatz, A new Hplc Solvent Delivery System, Techn. Note 88-2 (1988) by permission of Hewlett-Packard, Waldbronn, Germany. 

A modern solvent delivery system consists of one or more pumps, solvent reservoirs, and a degassing system. HPLC pumps can be categorized in several ways by flow range, driving mechanism, or blending method. A typical analytical pump has a flow range of 0.001-10 mL/min, which handles comfortably the flow rates required for most analytical work (e.g., 0.5-3 mL/min). Preparative pumps can have a flow range from 30 mL/min up to L/m. 

The assay was performed with a HPLC-system consisting of a Spectra-Physics (Spectra Physics, San Jose, CA 95134, USA) model SP8700 solvent delivery system used at a flow rate of l.Oml.min", a Kratos (Kratos Analytical Instruments, Ramsey, NJ 07446, USA) model 757 UV-detector, wavelength 260 nm, range 0.005 aufs, rise-time 1 second. Injections of extracts into a Zymark (Zymark Corporation Inc., Hopkinton, MA 01748, USA) Z 310 HPLC-injection station, equipped with an electrically controlled Rheodyne valve and a 20 pi sample loop, were performed by a Zymate II robot system. The Zymark Z 310 Analytical Instrument Interface was used to control the HPLC-injection station. 

An HPLC system, equipped with a Waters solvent delivery system (M-45), two PLgel 20 p,m Mixed-A columns (300 x 7.5 mm) with 20 p,m guard column (50 x 7.5 mm) (Polymer Laboratories, Amherst, MA) and a refractive index detector (model 2410) (Waters, Milford, MA), can be used to study the molecular size and size distribution (e.g. molecular weight and weight distribution) of starch. 

All of the fat-soluble vitamins, including provitamin carotenoids, exhibit some form of electrochemical activity. Both amperometry and coulometry have been applied to electrochemical detection. In amperometric detectors, only a small proportion (usually <20%) of the electroactive solute is reduced or oxidized at the surface of a glassy carbon or similar nonporous electrode in coulometric detectors, the solute is completely reduced or oxidized within the pores of a graphite electrode. The operation of an electrochemical detector requires a semiaqueous or alcoholic mobile phase to support the electrolyte needed to conduct a current. This restricts its use to reverse-phase HPLC (but not NARP) unless the electrolyte is added postcolumn. Electrochemical detection is incompatible with NARP chromatography, because the mobile phase is insufficiently polar to dissolve the electrolyte. A stringent requirement for electrochemical detection is that the solvent delivery system be virtually pulse-free. 

Instrumentation. The HPLC equipment consisted of two Model 740 solvent delivery systems combined with a Model 744 solvent programmer, Model 714 pressure monitor and a Model 755 sample injector (all from Spectra-Physics, Santa Clara, CA). The column used was 25 cm x 3 mm i.d. stainless steel tube packed with either Spherisorb silica 5 y or Spherisorb ODS 5 y. Detection was made with a Schoeffel Instrument Corporation (Westwood, NJ) Model SF 770 spectromonitor. Peak areas were measured by the cut and weight method. Radioactivity was measured by direct measurement in a Searle Model 1185 Automatic Gamma System. 

A hot water dynamic extraction device was constructed using the solubilization apparatus of Miller and Hawthorne (10) as a model and was used for pressurized extraction of milk thistle seed meal. Figure 2 shows a schematic diagram of the apparatus. Water, the extraction solvent, was pressurized and pumped using a Bio-Rad 2800 HPLC solvent delivery system (Hercules, C A) to an extraction cell housed in the oven of a Hewlett-Packard 5890 gas chromatography (GC) oven (Wilmington, DE). Before... 

In another publication (13) it was demonstrated that with proper maintenance and operating care each of two HPLC solvent delivery systems was capable of delivering smooth, precise flow rates of as low as a few microliters per minute. While not all chromatographers will desire this high degree of precision, it is clear that if people observe good laboratory practice,... 

Equipment for recycle operations differs from conventional HPLC equipment. For a recycle system to be useful, the extracolumn band spreading must be small relative to the band spreading of the column. This involves the solvent delivery system, transport tubing, and detector(s). Also, because a recycle system is a closed system with a finite volume, the operator must be aware that fast-moving materials could eventually overtake slower-moving materials and remix. To prevent peak overlap, a means must be provided to allow the operator to remove a portion of the sample components before overlap can occur. 

An HPLC system, shown schematically in Figure 2.1, consists of a solvent reservoir, which contains the eluent or mobile phse a pump, often called a solvent delivery system an injector through which the sample is introduced into the system without a drop in pressure or change in flow rate the analytical column, which is usually stainless steel and contains the solid packing or stationary phase and a suitable detector to monitor the eluent. 

While one of the most confusing steps for the new user of HPLC is deciding what equipment to order, an even more difficult and frustrating step occurs after the equipment has arrived and connections must be made between the solvent delivery system (the pump), the various columns, and the detector. For the new user quickly discovers that connections are not made with the more familiar, easy-to-use flexible plastic, but with stainless steel tubing, which cannot be cut with scissors or easily coupled with plastic connectors. Thus special tools must be used for cutting, and connections must be made with... 

Analysis of Phenolic Compounds. A Hewlett-Packard (Palo Alto, CA) Model 1090 HPLC System, was used to determine the levels of specific phenolic components. The HPLC system was equipped with a ternary solvent delivery system, a diode array UV-VIS detector, and HP ChemStation software for data collection and analysis. Full chromatographic traces were collected at 280, 520, 316, and 365 nm, and spectra were collected on peaks. The stationary phase was a Hewlett-Packard LiChrosphere C-18 coliram, 4mm X 250 mm, with 5 pM particle size packing. Operating conditions include an oven temperature of 40 C, injection volume of 25 pL, and flow rate of 0.5 mL/minute. The mefriod was based on a previously published method for phenolic components in wine (30) and used the modified solvent gradient shown in Table II. Solvent A was 50 mM dihydrogen ammonium phosphate, adjusted to pH 2.6 with orthophosphoric acid. Solvent... 

The HPLC analyses were performed on an Agilent Technology Model 1100 HPLC system equipped with a quaternary solvent delivery system, 1100 autosampler. 

HPLC was performed using Waters 600S solvent delivery system (Waters, Milford, MA, U.S.A.). 2487 UV dual channel detector of Waters was used and injector (20 fit sample loop) from Rheodyne. The data acquisition system was Millenium (Waters). Water filtered 1 Milipore ultra-pure water system (Milipore, Bedford, MA, USA). The wavelength was fixed at 254 nm and the experiment was performed at room temperature. The size of the analytical colunm packed by C g was lS0X4.6mm (Spm) (Alltech, USA). The mobile phase of 0.75% TFA in water and acetonitrile were used in this experiment. The flow rates of the mobile phase were fixed at I ml/min. The constant volume of 0(d, was injected. This experiment was implemented at room temperature. The gradient mode was employed to isolate peptides. The complete gradient condition was listed in Table I. 

Reversed phase HPLC was performed using fused silica columns and solvent delivery systems developed and built in our laboratory (5,6). All chromatographies were carried out on Vydac S itm C18 RP support with or without an SDS removal precolumn. SDS removal resin was obtained from Poly-LC Solvent A was 0.1 % trifluoroacetic acid (TFA) in water and solvent B was 0.07 % TFA, 90 % acetonitrile in water. Water was obtained from a Milli-Q system. Samples were eluted with a gradient from 2% to 92 % solvent B in 45 minutes unless otherwise noted As a standard, cytochrome C digested with Lys-C (CCKCD) was used The separation of the peptides was carried out with or without SDS. 

The essential components of an HPLC system are a solvent delivery system, a method of sample introduction, a column, a detector and an associated readout device (Fig. 32.16). 

Retention times tend to vary over time because of a number of causes, e.g., differences between batches of mobile phase, column performance and different columns, and ambient temperatures of laboratories. The RSD of retention time, especially important because it is used for peak identification, is influenced by 1) Pump flow 2) composition precision 3) mobile phase composition of solvent delivery systems and 4) column temperature. Imprecise retention time indicates problems within the HPLC system such as piston seals, check valves, etc. 

A modem HPLC system is shown schematically in Figure 2. The equipment consists of a high-pressure solvent delivery system, a sample auto injector, a separation column, a detector (often an UV or a DAD) a computer to control the system and display results. Many systems include an oven for temperature control of the column and a pre-column that protects the analytical column from impurities. The actual separation takes place in the column, which is packed with chemically modified 3.5-10 pm (often silica) particles. A mobile phase is pumped through the column with the high-pressure pump and the analytes in the injected sample are separated depending on their degree of interaction with the particles. A proper choice of stationary and mobile phase is essential to reach a desired separation. 

Gel permeation chromatography (gpc) was performed on a Waters GPC-3 with a model 600 solvent delivery system, a 730 data module, a variable wavelength ultraviolet detector (uv), and a refractive index detector (Rl). Calculations were made on the uv detector response with the wavelength set at 325 nm. Three j/Styragel columns of porosities 105, 104, and 103 A were used and calibrated with polystyrene standards. Injection size was 50-125//I of 0.05% solutions with a flow rate of 1.4 ml/min. The solvent was HPLC grade N-methyl pyrrolidone (NMP) obtained from Aldrich Chemical Co. buffered with 0.03 M LiBr and 0.03 M H3P04.(2, 3)... 

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