Sample capacity

The sample capacity is the maximum quantity of an analyte with which the phase can be loaded (Table 2.26). An overloaded column exhibits peak fronting, an asymmetrical peak which has a gradient on the front and a sharp slope on the back side. This effect can increase until a triangular peak shape is obtained, a so-called shark fin. Overloading occurs rapidly if a column of the wrong polarity is chosen. The capacity of a column depends on the internal diameter, the film thickness, polarity and the solubility of a substance in the phase. 

In addition to different column characteristics, one more factor must be taken into consideration in gas chromatography, namely the sample capacity. This is defined as the maximum permissible sample size that can be injected into a column without more than 10% loss of efficiency, and it is expressed as 

The reported values for a are on the order of 10 and common values for A for capillary columns are less than 0.1 mL (gas in normal conditions). 

This limitation indicated by the theory regarding the sample volume injected in a GC system imposes a serious problem when analyzing traces in a given sample. The detectors used in GC have limited sensitivity (see further), and an amount of sample below a certain limit cannot be detected. Therefore, a compromise should be chosen such that the sample should be small enough to be accommodated by the chromatographic column but sufficiently large for the detector sensitivity. 

An alternative for achieving a lower column load and enough analyte in the detector is to perform an additional separation before the analytes reach the analytical column. In this separation, part of the sample that is not of interest can be eliminated, and at the same time the important analytes can be kept. This preliminary separation can be done using bidimensional chromatography (see further), but simpler techniques are also reported, such as programmed temperature vaporization (PTV) injection, etc. 

The gas chromatographic separation in temperature gradient is affected by more than one factor [32]. Simultaneously there is variation in the gas flow, variation in the distribution constants, and variation in peak broadening. 

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Often, media of similar characteristics as the one used for prepacked columns (i.e., except for particle size) are obtainable, e.g., as preparative grade material, having particle sizes of typically 30-50 /j,m. This is required for large-scale gel filtration where the sample capacity of the prepacked columns is often insufficient. 

For best results, use the flow rate, injection volume, and column sample capacity guidelines in Table 3.11. Conditions outside these guidelines may be used, but poor resolution between proteins may result from extensive deviations from these guidelines. 

Methods of sample application. Due to the lower sample capacity of the H PTLC layer, the amount of sample applied to the layer is reduced. Typical sample volumes are 100-200 nL which give starting spots of only 1.0-1.5 mm diameter after developing the plate for a distance of 3-6 cm, compact separated spots are obtained giving detection limits about ten times better than in conventional TLC. A further advantage is that the compact starting spots allow an increase in the number of samples which may be applied to the HPTLC plate. 

Excellent open tubular columns may now be purchased, providing a number of stationary phases of differing polarity on WCOT and SCOT columns, and whose efficiency, greatly improved sample detectability, and thermal stability surpass those exhibited by packed columns their chief disadvantage is that they have a lower sample capacity than packed columns.65,66... 

To overcome the problems of relatively low sample capacity associated with SPME, a technique known as stir-bar sorptive extraction has been reported by Baltussen etal. A glass-coated magnetic stir bar was coated with 50-100 iL of PDMS. Sample extraction was performed by placing the stir bar in the sample with subsequent stirring for 30-120 min. After extraction, the stir bar was removed and analytes were thermally desorbed at 150-300 °C for 5 min for GC, or liquid desorbed for LC. Qualitative analysis of organochlorine residues in wine has been reported using a commercially available product known as Twister. ... 

Increasing the speed of analysis has always been an important goal for GC separations. All other parameters being equal, the time of GC separations can be decreased in a number of ways (1) shorten the column (2) increase the carrier gas flow rate (3) reduce the column film thickness (4) reduce the carrier gas viscosity (5) increase the column diameter and/or (6) heat the column more quickly. The trade-off for increased speed, however, is reduced sample capacity, higher detection limits, and/or worse separation efficiency. 

The applicable HPLC flow rate with ESI is lower than that with thermospray or APCI, usually below the O.SmLmin range. The typical flow rate is 0.10-0.20 mL min for ESI, which means that the effluent flow introduced into the electrospray must be reduced by splitting when using a conventional HPLC column (4.6-mm i.d. x 250 mm). Currently, narrower columns (e.g., 2.1-mm i.d.) and slower flow rates are commonly used to achieve the desirable flow rates. The advantage of this approach is that improved separation efficiency and faster separations are also achieved (at the cost of sample capacity). 

The sample capacity Q, arbitrarily defined as the maximum amount of a component that can be injected on a column giving a limited (10%) increase in peak width, is given by... 

The limited sample capacity and low carrier gas flow rates characteristic of open tubular column gas chromatography give rise to certain difl icultles in sample introduction. Direct sample... 

Totally porous particles of relatively large particle sizes were widely used in low pressure liquid chromatography for many years. These column packings had good sample capacity but only limited efficiency accompanied by long separation times, due to the large size and unfavorable size distribution of the particles and the presence of relatively deep pores within the particles through whick sample molecules diffused in and out of very slowly. 

It is difficult to compare separation techniques in any general way. Comparison may be based on the traditional figures of merit, such as resolution Rs (including column efficiency N, selectivity, retention, and peak capacity), chromatographic speed, sample capacity, sensitivity, detection and column impedance, as well as breadth of application. Usually a tradeoff between these attributes is found. Berger [26] has compared GC, pSFC, cSFC, LC and CE on the basis... 

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