Direct sample injection, solid phase extraction

Methods of analysis by direct injection in the column of sample were proposed (Lea et al., 1979 Nagel et al., 1979 Ong and Nagel, 1978 Wulf and Nagel, 1976 Roggero et al., 1989 Lamuela-Raventos and Waterhouse, 1994), but usually, prior to analysis, the different classes of compounds are fractionated on absorbent polymers such as polyamide, Sephadex LH20 or C18. The stationary phase C18 is also used for concentration and purification of the sample by solid phase extraction (SPE) prior to analysis. 

There are basically three methods of liquid sampling in GC direct sampling, solid-phase extraction and liquid extraction. The traditional method of treating liquid samples prior to GC injection is liquid-liquid extraction (LLE), but several alternative methods, which reduce or eliminate the use of solvents, are preferred nowadays, such as static and dynamic headspace (DHS) for volatile compounds and supercritical fluid extraction (SFE) and solid-phase extraction (SPE) for semivolatiles. The method chosen depends on concentration and nature of the substances of interest that are present in the liquid. Direct sampling is used when the substances to be assayed are major components of the liquid. The other two extraction procedures are used when the pertinent solutes are present in very low concentration. Modem automated on-line SPE-GC-MS is configured either for at-column conditions or rapid large-volume injection (RLVI). 

Other techniques to improve throughput are instrumentation based and may involve multiple HPLC systems. The simplest method involves the automated use of solid phase extraction cartridges for sample cleanup followed by direct injection into the mass spectrometer [114], Coupling of multiple HPLC systems to one mass spectrometer allows one column to equilibrate and separate while another column to flow into the mass spectrometer. Multiple HPLC systems may be configured such that the mass spectrometer is only exposed to each serial HPLC eluent as the analyte of interest is eluted [115,116]. Although multiple H P LC-based methods may increase throughput, they also typically decrease sensitivity and may confound data workup and interpretation. 

Section II covers the latest trends in reducing sample preparation time, including direct sample infusion/injection and on-line solid phase extraction (SPE). In Section III, we focus on newer trends in stationary phases and how these phases hope to offer different selectivities compared to current CIS-based phases. Section IV briefly provides a few observations on how new detectors are increasing the versatility of HPLC. Finally, in Section V we examine monolithic columns, small particles packed in short columns, high-temperature LC, ultra high-pressure LC, and parallel injection techniques. 

This particular fractionation step may be optional. Some samples can be directly injected after filtration (step 2) without solid-phase extraction. This technique, however, will improve the resolution of many of the HPLC polyphenolic peaks and will allow their analysis and identification. 

The sample preparation techniques described in the Basic and Alternate Protocols require 1 to 3 hr prior to sample injection into the HPLC. No additional time for sample cleanup is necessary if the sample is directly injected into the HPLC column without solid-phase extraction. 

Solvent extraction in combination with TD-GC-FID gives the opportunity to concentrate the extract directly on the adsorbent tube (solid phase extraction) by injection of the sample extract and purge off the solvent with for example, helium. Methanol extracts of house dust can be concentrated on adsorbent tubes by injecting up to 50 jllI and analyze the tubes by TD-GC (Kofoed-Sorensen and Clausen, 2004). 

Samples rarely come in a form that can be injected directly into the instrument some form of sample preparation usually is required. Sample preparation includes any manipulation of the sample prior to analysis, including techniques such as weighing, dilution, concentration, filtration, centrifugation, and liquid- or solid-phase extraction. Sample preparation can be performed either on-line or off-line, but it is usually performed offline. Off-line preparation can be time-consuming and tedious, and the more steps that are required, the more susceptible the analytical method is to operator error and irreproducibility. 

While it is wonderful to be able to inject neat samples directly, sample preparation can often improve selectivity and sensitivity. If the resolution is poor, the salt content of the sample too high, or the capillary fouls, consider a sample cleanup. This can include liquid-liquid extraction, solid-phase extraction, supercritical fluid extraction, protein precipitation, or dialysis, depending on the solutes and application [38]. The final sample diluent should be a solution that is CE-Mendly. That usually means low ionic strength compared to the BGE. 

Thormann et al. (1993) have published an overview of the strategies for using MEKC to monitor drugs in body fluids (serum, urine, saliva) they discuss buffer selection and sample preparation (direct injection, ultrafiltration, solid phase extraction. 

Determination of these compounds is carried out frequently in biological fluids. Analysis in urine requires a previous filtration to remove cells and other particulate matter then, the samples are diluted and directly injected onto the column. With cerebrospinal fluid, the samples are obtained by lumbar puncture each ahquot is centrifuged and decanted before analysis. Often in plasma or semm, some form of protein removal is needed because the presence of these compounds in injected samples can cause modifications of the column and bias in chromatographic results. Protein removal can be performed by various methods such as protein precipitation, ultrafiltra-tion, centrifugation, liquid-phase or solid-phase extraction, and column-switching techniques. 

Preliminary extraction of 5-HIAA may be used as an initial purification step before HPLC analysis. Organic solvents, anion-exchange resins, and other solid phase extraction procedures have aU been used. For many systems, direct injection of urine onto the analytical column is a common practice,and samples are often merely diluted with a buffer to protect the HPLC system from contamination. Methods that analyze 5-HIAA without prior sample cleanup rely on the selectivity of the HPLC separation combined with fluorescence or electrochemical detection to provide the requisite specificity. 

The overriding principle in many extractions of aqueous samples is for the analyte molecules to be as near neutral as possible by adjusting the pH of the sample matrix. This guiding principle applies to most liquid—liquid and reverse-phase solid-phase extractions. If a compound cannot be made electrically neutral through pH adjustment, then an alternate approach could be considered. A compound such as a zwitterion, which cannot be made electrically neutral could be isolated from the matrix using an approach which is insensitive to the ionization state of the molecule, such as direct injection, protein precipitation, microdialysis, or ultrafiltration. 

Three recently introduced on-line extraction techniques have been applied to biological samples. These techniques involve direct injection with restricted access media [11,12], turbulent flow chromatography [13], and on-line solid-phase extraction. 

The use of automation results in fast, easy, and reliable methods for SPE. There are a number of instruments available for totally automated SPE, as well as automated methods development of SPE, which span the spectrum in cost. In environmental applications, the choices of automation are limited, with only the Autotrace by Tekmar available for large-volume samples (1 L). In the area of on-line SPE-HPLC, nearly all of the workstations for SPE have the option for direct injection into the HPLC. For on-line SPE-GC/MS, there are several instruments and methods available (a modified PROSPEKT, Varian automated SPME, and the Hewlett-Packard solid-phase extraction workstation). For total automation including addition of internal standards, derivatiza-... 

Capella-Peiro et al. (28) used a 3 full factorial design to optimize the capillary zone electrophoresis (CZE) separation of a group of seven antihistamines (brompheniramine, chlorpheniramine, cyproheptadine, diphenhydramine, doxylamine, hydroxyzine, and loratadine). In this case, critical parameters such as pH (a concentration of 20 mM phosphate was kept constant in all the experiments) and the applied voltage were studied to evaluate their effect on the resolution and efficiency. Maximum response was achieved at pH 2.0 and an applied voltage of 5 kV. After a repeatability study to check the precision of the electrophoretic method, as well as a suitable calibration, the usefulness of this optimized method was demonstrated through the determination of the listed histamines in pharmaceuticals, urine, and serum samples (recoveries were in agreement with the stated contents). Urine samples were diluted and directly injected in the CE system, while serum samples were previously extracted by means of a solid-phase extraction (SPE) procedure. 

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