Injector Loop Sizing

An HPTC injector allows the introduction of a precise sample volume onto the column. A typical manual injector consists of a 6-port valve with a rotor, a sample loop and a needle port (Eigure 9). A sample solution is introduced into the sample loop using a 22-gauge blunt tip syringe in the TOAD position. The sample is then injected into the column by switching the valve to INJECT. The typical external sample loop size ranges from 6 pT to 2 mT. For many years, the Rheodyne 7125 injector was the industry-standard. In the early 1990s, it was replaced by the Rheodyne 7725 injector, which injects samples without momentary flow disruptions. ... 

Solvent volumes per run do not exceed the capacity of the syringe pump(s) because of the extremely low flow rates. Injection size is limited in these systems by incorporating a fixed-volume injector loop within the body of the... 

The sample loop size can be varied depending on the volume of sample. Typical sample loops range from 5 /A to 5 ml, and each must be manually removed before another can be put in place. The replacement of one loop size for another is one of the major disadvantages of the injector design. The accuracy of this type of injector varies with sample loop size, from about 5% for a 2-ml loop to as much as 30% for a 5-pl loop. 

Even with all these chromatographic parameters optimised, sensitivity may not be enough. The simplest way to increase sensitivity further is to inject more sample. Valve injectors are usually supplied with 20-pl loops but there are few assays for which 100-200 pi of sample cannot be injected simply by changing the loop size. A further way is to use minibore (1 mm or 2mm i.d.) or even microbore (< 1 mm) columns. Such columns give increases in sensitivity due to the narrower peaks eluted but this may reduce column loading volumes and with some equipment the extra sensitivity gained may be lost due to extra column band broadening. 

The TREF part of their cross fractionation system is housed in a heater block to allow the use of an injector loop and to heat the valving system needed to deliver the TREF fraction solution to the SEC. Column volume and sample size, solvent and concentration are directed by needs of the SEC analysis. They achieved a workable system with 15 x 0.8 cm colunm packed with 3.5 g of glass beads. The polymer (20 mg) was loaded hot as a 1% solution in o-dichlorobenzene. The cooling step caused some problems due to diffusion of polymer species outside the column. They solved this problem by fast cooling the solution (100 K/hour) but clearly there is concern about the effect of this on the quality of the TREF separation. A dissolution period of 10 minutes during the stepwise TREF was... 

A possible source of error when using reversed-phase systems is the adsorption of surfactants on the surfaces of the HPLC apparatus. For example, if the sample is injected as a purely aqueous solution, surfactant will build up in the metal injection valve depending on the volume of sample used to flush the loop. When the HPLC mobile phase is diverted through the valve, it will remove the adsorbed surfactant along with the measured sample volume. Thus, if varying volumes of sample are used to flush the injector, the peak size observed for a specific surfactant concentration will vary, even with the same loop size (93). This effect is minimized by dissolving the sample in the mobile phase. 

Non-Aqueous SEC Experiments. Non-aqueous SEC separations were carried out at ambient temperatures using two Varian MicroPak TSK GMH6 columns in series (7.5mm i.d. x 30cm each). This column is a mixed bed column containing pore sizes from 250 8 to 107 blended to ensure linearity of the molecular weight calibration curve. The mobile phase employed tetrahydrofuran at a flow rate of 1 ml/mln. Sample injection volumes were 50p)l using a Rheodyne 7126 manual loop Injector. 

Micro-injections in micro-flow and nano-flow systems are done with injectors in which the external sample loop is replaced with the internal fixed volume within the injector body. HPLC-on-a-chip systems also build the column into the injector body. The internal path within the injector body is abladed with a laser, packed with micro-packing material, and this serves as the separating media. The injector inlet is connected to the pumping system and the outlet to the detector. Sample is loaded into an internal loop in the load position, then injected onto the chip HPLC by turning the injector. Obviously, in a system like this sample size is very limited and the detector is usually a highly sensitive mass spectrometer. 

Sample introduction is a major hardware problem for SFC. The sample solvent composition and the injection pressure and temperature can all affect sample introduction. The high solute diffusion and lower viscosity which favor supercritical fluids over liquid mobile phases can cause problems in injection. Back-diffusion can occur, causing broad solvent peaks and poor solute peak shape. There can also be a complex phase behavior as well as a solubility phenomenon taking place due to the fact that one may have combinations of supercritical fluid (neat or mixed with sample solvent), a subcritical liquified gas, sample solvents, and solute present simultaneously in the injector and column head [2]. All of these can contribute individually to reproducibility problems in SFC. Both dynamic and timed split modes are used for sample introduction in capillary SFC. Dynamic split injectors have a microvalve and splitter assembly. The amount of injection is based on the size of a fused silica restrictor. In the timed split mode, the SFC column is directly connected to the injection valve. Highspeed pneumatics and electronics are used along with a standard injection valve and actuator. Rapid actuation of the valve from the load to the inject position and back occurs in milliseconds. In this mode, one can program the time of injection on a computer and thus control the amount of injection. In packed-column SFC, an injector similar to HPLC is used and whole loop is injected on the column. The valve is switched either manually or automatically through a remote injector port. The injection is done under pressure. 

Figure 4.11 (Continued) (B) The Rheodyne injector (Rheodyne, Inc.) in which the volume injected is determined by the sample loop. Sample loops can be obtained commercially in a number of fixed volumes or made to any size. (C) A 200-/xL loop.

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. 

The HPLC system comprised a Beckman 116 programmable solvent pump with a Beckman 168 photodiode detector—this was checked and data were processed with the Gold Nouveau software system (Beckman Coulter, Palo Alto, CA) and a Beckman 507 automatic injector with a 100.0-pL loop and a heating chamber for the columns. Analyses were carried out with an Ultrasphere ODS column (5.00-pm particle size, 15.0 cm x 4.60 mm internal diameter) purchased from Beckman Coulter. A guard column (2.00 cm x 2.00 mm internal diameter), packed with Sperisorb RP-18 (30.0 0.0 pm pellicular), was supplied by Upchurch Scientific (Oak Harbor, WA). 

Size Exclusion Chromatography. Exclusion chromatography was carried out on an apparatus comprised of a Minipump (Milton Roy), a model 7012 injector (Rheodyne) equipped with a 100-pl loop, an R401 differential refractometer (Waters), and a Model 153 UV detector, X,=254 nm (Altex). A Superose-6 column (30 cm x 1 cm OD)(Pharmacia) was eluted at 0.53 ml/min.. Column efficiency, determined with D2O, was at least 12,000 plates/meter. 

Samples can be introduced manually into the valve with a syringe to fill the sample loop. Automated sampling valves are routinely used today in which samples are taken from an autosampler for unattended operation. The major limitation of valve injectors is that the sample size is fixed, and the loop must be changed in order to vary the injected sample size. There are automated motor-driven adjustable syringes that provide enough pressure to inject the sample past a check valve that prevents backflow. 

The 7(M0 Sanvle Irriection Vatve. Here s s 6-port sample injection valve with a removable sample loop and 7000 psi pressure rating. Size, to ii to 2.0 ml The 7120 Syringe Load Sample Injector. Fil loops conventionally or In the partial loop variable vDlume mode with only Q5 fi sample loss. 

The HPLC analyses were conducted with a Water 600 Pump apparatus. This apparatus was equipped with a quaternary solvent delivery system, a Rheodyne injector with 20qL sample loop and a UV detector Waters 486 Tunable which was fixed at 280 nm. Throughout this study. Alltech Intertsil ODS-5 C18 reversed phase column (150 mm, 4.6 mm, 5qm particle size) was used. The flow rate of the mobile phase was of 1 mL / min and the gradient elution was adapted from (Nakatani et al., 2000 Bouayed et al, 2007). The solvent composition and the gradient elution program are reported in the table 1. 

These measurements clearly show that convective flow Is the major origin of the dispersion observed at the output of a loop Injector. However, these measurements were made In a time domain which Is close to that where dispersion by diffusion should start to become apparent according to hydrodynamic theory. This Is apparent In the data obtained closest to the wall where the dispersion Is greatest. Solution linear velocity Is lower In this region giving greater time for diffusion. The small size of the detector allows the radial concentrations to be probed with little perturbation of the stream. This Is evidenced by the ability to reconstruct the bulk behavior from the individual radial measuresients. 

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