Dilute samples,injection problems

Carryover. Small amounts of analyte may get carried over from the previous injection and contaminate the next sample to be injected [10]. The carryover will affect the accurate quantitation of the subsequent sample. The problem is more serious when a dilute sample is injected after a concentrated sample. To avoid cross-contamination from the preceding sample injection, all the parts in the injector that come into contact with the sample (the injection loop, the injection needle, and the needle seat) have to be cleaned effectively after the injection. The carryover can be evaluated by injecting a blank after a sample that contains a high concentration of analyte. The response of the analyte found in the blank sample expressed as a percentage of the response of the concentrated sample can be used to determine the level of carryover. Caffeine can be used for the system carryover test for assessing the performance of an injector and serves as a common standard for comparing the performance of different injectors. 

Mass overload occurs when the stationary phase does not have the capacity to retain the amount of sample injected. This can occur even for small injection volumes if the concentration of sample is high enough. This results in a characteristic shark-fin peak shape, where peak tailing starts from the peak s apex. For example, in order to obtain sufficient sensitivity, analytes with weak UV molar absorptivity may require a large enough amount of sample to be injected that the stationary phase becomes overloaded. Injecting less amount of sample, either by a smaller injection volume or by diluting the sample, can solve the problem of mass overload. However, sensitivity will decrease in this case. 

Headspace GLC analysis is a method used to monitor a vapour over a polymeric matrix. It is a very effective technique, but may require more time and effort than direct injection. This method can be performed manually, when a vial containing the monomer is heated, an equilibrium is established, for volatile compounds between the sample and the headspace above it. Because no dissolution step is required, sample viscosity problems and loss of response due to dilution are eliminated. Automated headspace analysis units are available from instrument manufacturers, as well as multiple extraction systems. Any analytically useful headspace method must obey Henry s law ... 

The Micromeritics Instrument Corporation is currently developing an integrated hydrodynamic chromatograph. Problems with development and/or manufacture have resulted in some delays, but a prototype should be available for purchase late in 1982. The instrument will apparently contain its own microprocessor for control of instrument operation, and also for deconvolution of the data to account for axial dispersion. Elution times are not available at this time, but if they can be kept to a minimum, and if sampling, dilution and injection functions can be automated, this instrument may provide highly accurate, on-line particle size distribution data for reactor control. 

The elution of the organic compounds collected involves extraction by a solvent (displacement) or thermal desorption. Pentane, CS2 and benzyl alcohol are generally used as extraction solvents. CS2 is very suitable for activated charcoal, but cannot be used with polymeric materials, such as Tenax or Amberlite XAD, because decomposition occurs. As a result of displacement with solvents, the sample is extensively diluted, which can lead to problems with the detection limits on mass spectrometric detection. With solvents additional contamination can occur. The extracts are usually applied as solutions. The readily automated static headspace technique can also be used for sample injection. This procedure has also proved to be effective for desorption using polar solvents, such as benzyl alcohol or ethylene glycol monophenyl ether (1% solution in water, Krebs, 1991). 

As in tic, another method to vaUdate a chiral separation is to collect the individual peaks and subject them to some type of optical spectroscopy, such as, circular dichroism or optical rotary dispersion. Enantiomers have mirror image spectra (eg, the negative maxima for one enantiomer corresponds to the positive maxima for the other enantiomer). One problem with this approach is that the analytes are diluted in the mobile phase. Thus, the sample must be injected several times. The individual peaks must be collected and subsequently concentrated to obtain adequate concentrations for spectral analysis. 

Additionally, the inj ected matrix must also be miscible with the solvents used in the separations. For normal phase mode separations, all water must be removed from the injected matrix. Since many of the complex matrixes, such as plasma, urine, and other biological fluids contain a large amount of water, this requires more time consuming sample preparation. However, water can be injected into a polar organic or reverse phase mode separation. Even within the same mode, mobile phases that are very different can cause large disturbances in the baseline. Oda et al., (1991) solved this problem by inserting a dilution tube followed by a trap column in order to dilute the mobile phase used on the achiral column. Following the dilution tube, a trap column was used to reconcentrate the analyte of interest before the enantiomeric separation. 

Matrix effect is a phrase normally used to describe the effect of some portion of a sample matrix that causes erroneous assay results if care is not taken to avoid the problem or correct for it by some mechanism. The most common matrix effects are those that result in ion suppression and subsequent false negative results. Ion enhancement may lead to false positive results.126 127 Several reports about matrix effects include suggestions on what can cause them and how to avoid them.126-147 While various ways to detect matrix effects have been reported, Matuszewski et al.140 described a clear way to measure the matrix effect (ME) for an analyte, recovery (RE) from the extraction procedure, and overall process efficiency (PE) of a procedure. Their method is to prepare three sets of samples and assay them using the planned HPLC/MS/MS method. The first set is the neat solution standards diluted into the mobile phase before injection to obtain the A results. The second set is the analyte spiked into the blank plasma extract (after extraction) to obtain the B results. The third set is the analyte spiked into the blank plasma before the extraction step (C results) these samples are extracted and assayed along with the two other sets. The three data sets allow for the following calculations ... 

A schematic diagram of the automatic system is shown in Fig. 4.7a. The sample -to-gas-chromatograph interface and requirements for the injection of a hquid mixture onto a gas chromatographic column from a flowing stream present the greatest problem, in contrast to sample transfer and dilution techniques in automatic analysis, which are well documented. 

The handling and disposal problems associated with the use of liquid solvent extractors have resulted in increased attention to the separation and preconcentration of organic compounds in water by collection in synthetic polymers followed by elution with an organic solvent (2). For example, selective collection and concentration of organic bases on methylacrylic ester resin from dilute water samples have been reported (3). Such collection techniques are especially well-suited to flow-injection measurement techniques. In this study, ionizable organic analytes such as salicylic acid and 8-hydroxyquinoline (oxine) were extracted into a polymer and then back extracted by an aqueous solution. Amperometric measurement using a flow-injection technique was employed to monitor the process. 

One way to avoid the nonlinear problem is to dilute the standard and unknown with a compatible solvent that is fully resolved chromatographically from all of the sample components. These dilutions need not be accurately made or need not be the same for the standard and unknown. Good practice dictates that they be approximately the same for each. This is merely a technique for injecting a smaller amount of the standard and sample into the chromatograph. Since the calculations do not involve sample size, this dilution is not a factor the solvent and any solvent impurity peaks are not measured and are not to be considered in the calculations. 

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