Sample injection in HPLC

Sample injection in hplc is a more critical operation than in gc. Samples may be injected either by syringe or with a valve injector although the former is now rarely used. Valves, which can be used at pressures up to about 7 000 psi (500 bar), give very reproducible results for replicate injections (<0.2% relative precision) and are therefore ideal for quantitative work (p. 129). They consist of a stainless steel body and rotating central block into which are cut grooves to channel the mobile phase. from the pump to the column (figure 4.27). The sample is loaded into a stainless steel loop incorporated... 

A precision injection device is required to minimize sample dispersion and keep the sample volume and length of sample zone reproducible. This is normally a rotary valve similar to that used for injection in HPLC. Exact timing from sample injection to detection is critical because of rapidly occurring reactions which are monitored before they reach completion. This demands a constant flow rate with low amplitude pulsing, normally achieved by a peristaltic... 

Injectors introduce the sample into the mobile phase under high pressure. There are several approaches to injection in HPLC, such as syringe injection via septum, a combination of a septum and syringe or a valve injection. Valve injection is the method of preference in up-to-date HPLC instrumentation. 

The study of the precision of a method is often the most time and resource consuming part of a method validation program, particularly for methods that are developed for multiple users. The precision is a measure of the random bias of the method. It has contributions fi om the repeatability of various steps in the analytical method, such as sample preparation and sample injection for HPLC [5-9], and from reproducibility of the whole analytical method fiom analyst to analyst, fiom instrument to instrument and fiom laboratory to laboratory. As a reproducibility study requires a large commitment of time and resources it is reasonable to ensure the overall ruggedness of the method before it is embarked upon. 

The need for unattended and precise sample injection for HPLC has lead to development of a wide variety of automated sample injection devices. Autosamplers function in essentially the same way as manual injectors, except that the sample is introduced automatically from a sample vial held in a carousel or an X-Y grid (Fig. 3.15). The carousel format provides a reliable and rapid means of moving samples past an injection station, whereas the XY grid format allows a convenient random access configuration. 

The most common method of sample introduction in HPLC is via a rotary valve, e.g. a Rheodyne valve. A schematic diagram of a rotary valve is shown in Fig. 32.17. In the load position, the sample is introduced via a syringe to fill an external loop of volume 5, 10 or 20 L. While this occurs, the mobile phase passes through the valve to the column. In the inject position, the valve is rotated so that the mobile phase is diverted through the... 

DCP chromatograms for these very same animal feed premix samples were identical to a standard chromatogram of both Se species, as in Fig. 9.15, and clearly demonstrated the presence of one or both Se species, sample dependent. A blank injection in HPLC-DCI of just the sample matrix without spiked selenium species, showed a perfectly flat baseline with absolutely no plasma responses. There was little or no baseline disturbance via DCP detection, as opposed to Uy and the only species peaks observed were at the expected/correct retention times for Se(IV) and/or Se(VI). This was one of the clearest demonstrations of how and when DCP detection provides adequate sensitivity, detection limits, and superb anal d e selectivity as well as specificity when interfaced with chromatography. 

This, of course, is the concentration of fluoranthene in the sample injected into the HPLC. The concentration of fluoranthene in the soil is... 

Injecting the Sample The mechanism by which samples are introduced in capillary electrophoresis is quite different from that used in GC or HPLC. Two types of injection are commonly used hydrodynamic injection and electrokinetic injection. In both cases the capillary tube is filled with buffer solution. One end of the capillary tube is placed in the destination reservoir, and the other is placed in the sample vial. 

A flow scheme for the basic form of ion chromatography is shown in Fig. 7.3, which illustrates the requirements for simple anion analysis. The instrumentation used in IC does not differ significantly from that used in HPLC and the reader is referred to Chapter 8 for details of the types of pump and sample injection system employed. A brief account is given here, however, of the nature of the separator and suppressor columns and of the detectors used in ion chromatography. 

In contrast, in HPLC assays the chromatographic separation and the integration of the resulting analyte peak normally are just as or even more error-prone than is the preparation of the solutions here it would be acceptable to simply reinject the same sample solution in order to obtain a quasi-independent measurement. Two independent weighings and duplicate injection for each solution is a commonly applied rule. 

A stream of ethylene is fed into the reactor by use of quaternary LC pumps and subsequently dissolved in a 1.90 ml h toluene stream [1]. Ethylene is handled at 60 °C, well above the critical temperature. Catalyst additions are fed via HPLC-type sample injection valves. Various combinations of precatalysts and activators were sampled and loaded by an autoinjector. Catalyst solutions typically were diluted 20-fold within the micro reactor. 

The modem HPLC system is a very powerful analytical tool that can provide very accurate and precise analytical results. The sample injection volume tends to be a minor source of variation, although fixed-loop detectors must be flushed with many times their volume in sample to attain high precision. Assuming adequate peak resolution, fluorimetric, electrochemical, and UV detectors make it possible to detect impurities to parts per billion and to quantitate impurities to parts per thousand or, in favorable cases, to parts per million. The major sources of error in quantitation are sample collection and preparation. Detector response and details of the choice of chromatographic method may also be sources of error. 

Inman, E. L. and Tenbarge, H. J., High-low chromatography estimating impurities in HPLC using a pair of sample injections, /. Chromatogr. Sci., 26, 89,... 

The basic SFC system comprises a mobile phase delivery system, an injector (as in HPLC), oven, restrictor, detector and a control/data system. In SFC the mobile phase is supplied to the LC pump where the pressure of the fluid is raised above the critical pressure. Pressure control is the primary variable in SFC. In SFC temperature is also important, but more as a supplementary parameter to pressure programming. Samples are introduced into the fluid stream via an LC injection valve and separated on a column placed in a GC oven thermostatted above the critical temperature of the mobile phase. A postcolumn restrictor ensures that the fluid is maintained above its critical pressure throughout the separation process. Detectors positioned either before or after the postcolumn restrictor monitor analytes eluting from the column. The key feature differentiating SFC from conventional techniques is the use of the significantly elevated pressure at the column outlet. This allows not only to use mobile phases that are either impossible or impractical under conventional LC and GC conditions but also to use more ordinary... 

Recently a decreased level of CE activity has been noticed with a shift of attention towards other separation techniques such as electrochromatography. CE is apparently not more frequently used partly because of early instrumental problems associated with lower sensitivity, sample injection, and lack of precision and reliability compared with HPLC. CE has slumped in many application areas with relatively few accepted routine methods and few manufacturers in the market place. While the slow acceptance of electrokinetic separations in polymer analysis has been attributed to conservatism [905], it is more likely that as yet no unique information has been generated in this area or eventually only the same information has been gathered in a more efficient manner than by conventional means. The applications of CE have recently been reviewed [949,950] metal ion determination by CE was specifically addressed by Pacakova et al. [951]. 

Figure 7.5 Schematic representation of a coupled SFE-HPLC system employing a recirculating extraction manifold interfaced to HPLC via a sample injection valve. After Lynch [54]. Reprinted from T. Lynch, in Chromatography in the Petroleum Industry (E.R. Adlard, ed.), J. Chromatography Library, 56, 269-303, Copyright (1995), with permission from Elsevier...

The earliest injection method in hplc used a technique borrowed from gc in which a microlitre syringe was employed to inject the sample through a self-sealing rubber septum held in an injection unit at the top of the column. In another method, (stopped flow), the flow of mobile phase through the column was halted and when... 

The loss of about 7% of the lst-D effluent caused by a 2-s injection in a 30-s operation cycle, which could cause up to 20% loss of a peak in the most unfavorable case, or the narrowest peak at the beginning, can be avoided by using two six-port valves each having a sample loop (Fig. 7.6b) an alternative system uses a 10-port valve with two holding loops. The loops hold the effluent of the lst-D alternately for 30 s during a complete separation cycle on the 2nd-D column to effect comprehensive 2D HPLC. 

The loop for the 2nd-D was loaded with the effluent of the 1 st-D at 50 pL/min for 1 min 58 s, and then the injection valve was turned to inject the 100 pL fraction for 2 s onto the 2nd-D HPLC. The flow rate was 5 mL/min, and the valve was turned back for the next loading, resulting in fractionation of the lst-D every 2 min. In this case less than 2% of the effluent from the 1 st-D was wasted during sample injection. The 2nd-D effluent eluted at 5 mL/min from the 2nd-D column, passed through a UV detector, and then was split by using a T-joint at approximately a 1/140 split ratio, resulting in a flow rate of ca. 36 pL/min going into the spray capillary for ESI-TOF-MS detection. 

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