Sample quantity, limitations

There will be many times when the quantity of sample is limited. While it is best to use the 92.9 cm" (0.1 ft") area leaf in order to minimize edge effects and improve accuracy, when the sample volume is limited it is much better to have several data points with a smaller leaf than onl one or two using the larger leaf. Data from leaves as small as 23.2 cm" (0.025 ft") are reasonably accurate and can be used to scale up to commercially sized units. However, it is usually prudent to employ a more conservative scale-up factor. 

No mention of throughput was made. Measurement of pKa by UV after separation by CE has been described for acids [41] and bases [42], and while this method only requires small sample quantities, the rate of throughput is currently about 20 samples per day [23, 43], A rate-limiting step in the CE method is the need to make separate experimental runs at different buffered pH values in order to determine the relative concentration of ionized species at each pH. 

In many applications, such as the analysis of mercury in open ocean seawater, where the mercury concentrations can be as small as 10 ng/1 [468,472-476], a preconcentration stage is generally necessary. A preliminary concentration step may separate mercury from interfering substances, and the lowered detection limits attained are most desirable when sample quantity is limited. Concentration of mercury prior to measurement has been commonly achieved either by amalgamation on a noble-metal metal [460,467, 469,472], or by dithizone extraction [462,472,475] or extraction with sodium diethyldithiocarbamate [475]. Preconcentration and separation of mercury has also been accomplished using a cold trap at the temperature of liquid nitrogen. 

Smaller diameter probes reduce sample volumes from 500 to 600 pi typical with a 5 mm probe down to 120-160 pi with a 3 mm tube. By reducing the sample volume, the relative concentration of the sample can be correspondingly increased for non-solubility limited samples. This dramatically reduces data acquisition times when more abundant samples are available or sample quantity requirements when dealing with scarce samples. At present, the smallest commercially available NMR tubes have a diameter of 1.0 mm and allow the acquisition of heteronuclear shift correlation experiments on samples as small as 1 pg of material, for example in the case of the small drug molecule, ibu-profen [5]. In addition to conventional tube-based NMR probes, there are also a number of other types of small volume NMR probes and flow probes commercially available [6]. Here again, the primary application of these probes is the reduction of sample requirements to facilitate the structural characterization of mass limited samples. Overall, many probe options are available to optimize the NMR hardware configuration for the type and amount of sample, its solubility, the nucleus to be detected as well as the type and number of experiments to be run. 

Classical laboratory, manual methods conducted on a macro-scale where sample quantities arc in the range of grams and several milliliters. These are the techniques that developed from the earliest investigations of chemistry and which remain effective for teaching the fundamentals of analysis. However, these methods continue to be widely used in industry and research, particularly where there is alarge variety of analytical work to be performed. The equipment, essentially composed of analytical balances and laboratory glassware, tends to be of a universal nature and particularly where budgets for apparatus are limited, the relative modest cost of such equipment is attractive. 

When sample quantity is absolutely limited, the NMR measurements can be made first, followed by moisture determination by the reference method on one part of the sample and oil determination on the other. Later, the reference moisture and oil values can be entered into the calibration file for correlating NMR signal/gram to the observed moisture and oil by the reference method. 

Chemists now routinely use open-access LC/MS in the same way that they previously used TLC to monitor reaction mixtures for the desired product and to optimize reaction conditions. In practice, medicinal chemists require only molecular mass data and are comfortable with a variety of ionization methods to obtain this information. However, confidence in the actual method and procedure is a requisite. Today, molecular mass measurement has quickly become a preferred means of structure confirmation over NMR and IR during the early stages of synthetic chemistry activities, where sample quantities are limited. 

Analytical technique Non-destructive analysis possible (conservation of the sample after analysis) Sample quantity required Sample preparation Analysis type Spatial resolution Limit of detection... 

Detection limit the smallest sample quantity that yields a signal that can be distinguished from the background noise. See also sensitivity. 

Traditionally, the impurities are isolated and purified by off-line HPLC and then characterized by using FT-IR, NMR, MS, and X-ray crystallography, among others. The main limitation associated with this approach is that relatively large sample quantities are needed for analysis, and the process can be very labor-intense. In contrast, LC/MS and LC/MS/MS are highly sensitive techniques requiring typically less than Ipg of material for analysis. In certain cases, if the impurities are found at very low levels in the drug substance, extraction procedures are used to concentrate them to detectable levels. 

Recently, NMR spectrometers directly coupled with LC systems have become commercially available. Spectra can be acquired in either of two modes, continuous or stopped flow. In continuous flow mode the spectrum is acquired as the analyte flows through the cell. This method suffers from low sensitivity since the analyte may be present in the cell for only a brief period of time, but it has the advantage of continuous monitoring of the LC peaks without interruption. Fig. 12A shows a contour plot of the continuous flow NMR analysis of a mixture of vitamin A acetate isomers.Fig. 12B shows the spectra taken from slices through the contour plot. These plots highlight the olefinic region of the spectra which provided ample information for the identification of each of the isomers. With very limited sample quantities, the more common method of LC-NMR analysis is stopped flow. Here the analyte peak is parked in the flow cell so any of the standard NMR experiments can be run. 

Size-exclusion chromatography [9] with a coupled molecular-weight-sensitive detection is a simple convenient method for characterizing dendrimers for which limited sample quantities are available. The polyether dendrimers increase in hydrodynamic radius approximately linearly with generation and have a characteristic maximum in viscosity. These properties distinguish these dendrimers from completely collapsed, globular structures. The experimental data also indicate that these structures are extended to approximately two-thirds of the theoretical, fully extended length. 

Several probe sizes are commercially available, and if one intends to do impurity and degradant characterizations routinely, it is important to have a representative range of sizes. The probe size is expressed in terms of the diameter of the sample tube for which it is designed. A common size is a 5-mm probe. This is an appropriate choice if one is not limited by sample quantity. The sample size is ideally 25-50 mg in 0.5-0.75 mL of solvent, although as little as 1 mg of material can be used. Obviously, increasing the sample concentration will reduce the experiment time required to obtain adequate signal-to-noise ratios. 

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