Loop injector, HPLC

One advantage of an HPLC analysis is that a loop injector often eliminates the need for an internal standard. Why is an internal standard used in this analysis What assumption(s) must we make about the internal standard ... 

As mentioned previously, introducing the sample to the flowing mobile phase at the head of the column is a special problem in HPLC due to the high pressure of the system and the fact that the liquid mobile phase may chemically attack a rubber septum. For these reasons, the use of the so-called loop injector is the most common method for sample introduction. 

FIGURE 13.7 The loop injector for HPLC, (a) the load positon—the sample is loaded into the loop via a syringe at atmospheric pressure, and (b) the inject position—the mobile phase sweeps the contents of the loop onto the column. 

What are some options that are available if an analyst wants to inject a volume that is smaller than the volume of a sample loop installed on an HPLC loop injector ... 

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.)...

Figure 21-23. Flow scheme of an on-line HPLC system developed in-house (1, mobile phase 2, HPLC pump 3, heat exchanger 4, injector with 2-p.L loop 5, HPLC-column 6, heating oven 7, UV cell 8, LFV lamp 9, UV detector 10, reference sample 11, restrictor 12, valves).

The solution as mobile phase (imidazole lOOmmol/L in the Tricine buffer 50 mmol/L, pH 9.4) was flowed at 0.1 mL/min using HPLC pump to the flow cell reactor via inlet connecting tube, and drained via a outlet tube. H2O2 specimen (20 juL) was injected with a loop injector (7125, JASCO). The reaction temperature for CL was room temperature. Light from the flow-through chip was detected after the dark frame reduction with the CCD-CL monitor. 

The absolute response factor (not to be confused with the partition coefficient), is not an intrinsic parameter of the compound since it depends upon the tuning of the chromatograph. To calculate the response factor AT, according to expression 4.5, it is essential that both the area A, and the mass m, of compound i injected on the column, are known. However, this mass is difficult to determine with precision since it relies simultaneously upon the syringe, upon the injector type (in GC), or upon the injection loop (in HPLC). This is why most chromatographic methods utilized for quantitative analyses, whether pre-programmed into an integrated recorder or in the multiplicity of available software, do not make use of the absolute response factors, K. ... 

The technique has many variations, which Koenigbauer and Majors (34) classified into four types direct transfer, indirect transfer, reversed transfer, and loop transfer. The analyte fraction of the first column eluate can be diverted directly to a second HPLC column (direct transfer), or eluted from column 1 to column 2 with a step gradient to a stronger mobile phase (indirect transfer), or eluted into a loop injector prior to the second column (loop transfer). The combination of the two columns provides sufficient clean-up for a specific analysis. With reversed transfer, the analyte is concentrated on the head of the first column and, after the sample interferences are passed through the first column, the analyte is transferred to the second column by reversing the flow through the first column. 

The HPLC was a Du Pont 8800 quaternary solvent system with a Hewlett-Packard (HP) 1040A photodiode array detector (5-8). Flexible disks were used for data storage, and postrun data evaluation was performed by the detector s computer (HP 85). Samples were injected with a loop injector 10 p,L was used for analytical scale, and 200 jlL was used for preparative scale. Spectra were run on a spectrophotometer (Perkin-Elmer Lambda 3) for static absorbance and on a spectrofluo-rometer (Perkin-Elmer MPF-66) for fluorescence. Field-ionization mass spectra were obtained at a resolution of 45,000 (corresponding to a 0.01-daltons error for a mass of 450). 

Rotary sample loop injector Figure 13.6 illustrates the most common design of HPLC injector. To introduce the sample as in GC with a syringe through a... 

The method of characterization by high-performance liquid chromatography [HPLC] is as follows 25 mg of hemicellulose was put into 50 mL volumetric flask and pure water was added to the mark. The mixture was shaken and then filtered [first few milliliters of filtrate was discarded]. The solution was then filtered through a membrane filter of 0.2 pm cellulose nitrate. Then about 100 mL solution was injected into the HPLC system via a loop injector with a 20 mL, using an isocratic elution system with distilled water with a mobile phase, flow rate 0.8 mL/min. The detection was made using a UV detector at a wavelength of 280 nm [34]. 

The way most TE columns are used with a valve-loop injector is that the sample solution first flows in one direction for capture, and then is backflushed onto the analytical column. This process of backflushing can also push particles captured on the inlet frit back onto the analytical column therefore, this is another reason for filtration/centrifugation of samples before introducing them onto the TE column. Any in-line filter can also prevent particulates from migrating to places in the HPLC system where they can cause problems. 

Loop Injectors for High Performance Liquid Chromatography (HPLC)... 

This final working equation is a better situation than that summarized in Equation [8.64f] since, although the various volumes still appear explicitly, the ratio of injection volumes (v /V) is likely unity with little or no uncertainty if an HPLC loop injector is used. Moreover, we now must assume only Fa = Fa " rather than Fj = 1 the only reason why Fa " might not be exactly equal to Fa is that the extraction efficiency Fa for the native analyte in the analytical sample might be less that for the spiked-in standard analyte (Fa ) a result of occlusion effects of some kind, as can happen with some samples (Boyd 1996, Ke 2000). 

The electrospray ionization tandem mass spectrometer was obtained from Finni-gan MAT. Pumps for HPLC were purchased from Applied Biosystems (Cat. No. 140B, dual-syringe pump), and a sample injector was obtained from Rheodyne (Cat. No. 8125, 5- 1 loop injector). 

In the case of capillary columns, finding appropriate solutions for sample introduction is much more challenging than with packed columns. Since packed columns have dimensions similar to normal HPLC, optimal sample introduction can be achieved with ordinary sample loop injectors (see Section 12.2.4.2). 

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