Retention factor sample preparation

Principles and Characteristics Although early published methods using SPE for sample preparation avoided use of GC because of the reported lack of cleanliness of the extraction device, SPE-GC is now a mature technique. Off-line SPE-GC is well documented [62,63] but less attractive, mainly in terms of analyte detectability (only an aliquot of the extract is injected into the chromatograph), precision, miniaturisation and automation, and solvent consumption. The interface of SPE with GC consists of a transfer capillary introduced into a retention gap via an on-column injector. Automated SPE may be interfaced to GC-MS using a PTV injector for large-volume injection [64]. LVI actually is the basic and critical step in any SPE-to-GC transfer of analytes. Suitable solvents for LVI-GC include pentane, hexane, methyl- and ethylacetate, and diethyl or methyl-f-butyl ether. Large-volume PTV permits injection of some 100 iL of sample extract, a 100-fold increase compared to conventional GC injection. Consequently, detection limits can be improved by a factor of 100, without... 

Preparative-scale chromatography relies on a compromise between three variables (cf. Figure 1) (i) component resolution (determined by selectivity, efficiency and retention factor), (ii) speed of analysis and (iii) column sample capacity (Pescar, 1971). Any two of the desired goals may be realized only at the expense of the third. If a large amount of sample is required in a short time, resolution must be high. If resolution is insufficient, either the column load is limited or the time required for separation is long. 

Different compounds have variable ionization intensities, which are further affected by factors such as chromatographic retention times and suppression by compounds present in the analyte fluid and sample matrix, so it is essential to use isotope-labeled internal standards, which are physicochemically identical to the molecule of interest rather than structural analogs, to normalize for effects that can lead to erroneous quantification. Isotope-labeled standards also account for any differences arising out of sample preparation and absorptive loss due to selective binding on surfaces during chromatography (32). 

In the analytical procedure, an accurately measured aliquot of the product is diluted Avith a diluent (normally the mobile phase) and the resulting sample solution is injected into the HPLC. Because the majority of injectable pharmaceuticals are clear solutions, typically a simple dilution step is all that is needed for sample preparation. However, if the parenteral product is an emulsion or a suspension, appropriate steps must be taken to dissolve the product to achieve a clear solution (ultrasonication, filtration, etc.). For the assay procedure, the sample concentration chosen should be such that the peak areas obtained from multiple injections from the same sample are reproducible with minimum variance (<2% relative standard deviation). Peak shape and retention time also play important roles in the precision of the assay. A tailing factor less than 1.5 and a capacity factor less than 10 for the active peak are generally required for a good analytical method. A reference standard solution having the same concentration and using the same diluent as the sample solution is prepared. 

The effect of these factors on the adsorption isotherm may be elucidated by reference to specific examples. In the case of the isotherm of Fig. 5.17(a), the nonporous silica had not been re-heated after preparation, but had been exposed to near-saturated water vapour to ensure complete hydroxylation. The isotherm is of Type II and is completely reversible. On the sample outgassed at 1000°C (Fig. 5.17(h)) the isotherm is quite different the adsorption branch is very close to Type III, and there is extrensive hysteresis extending over the whole isotherm, with considerable retention of adsorbate on outgassing at 25°C at the end of the run. 

Internal standards at a known concentration are added to the sample after its preparation but prior to analysis to check for GC retention-time accuracy and response stability. If the internal standard responses are in error by more than a factor of two, the analysis must be stopped and the initial calibration repeated. Only if all the criteria have been met can sample analysis begin. 

Basic Protocol 2 takes about an hour to prepare a sample, but by staggering the start of preparation, 2 to 3 samples/hour can be prepared. The limiting factor is getting them analyzed before they degrade, since the analysis time is about 30 min between injections. Remember that it will likely be necessary to prepare a concord grape standard and/or a strawberry standard periodically to confirm retention times. 

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