Sample Volume Injected

In industrial-scale chromatography, one very desirable aspect is the ability to handle large amounts of samples, without any increase in Hs. For sample volumes of 

In industrial-scale chromatography, one very desirable aspect is the ability to handle large amounts of samples, without any increase in Hs. For sample volumes of up to a few percent of the total column volume, Hs remains constant, but it then increases with the sample volume. It has been reported that the effect of sample volume on Hs should rather be treated as the effect of the sample injection time to (s) [7]- 

---

Sample volume injected (Vi) 2 xL Final solution volume (VEnd ) 4mL Sample weight (G) 20 g Detected amount (W)... 

Sample volume injected 2 aL Final solution volume 2 mL Sample weight 50 g... 

Sample volume injected 10 xL Final solution volume 10 mL Sample weight 5 g... 

Sample volume injected 20 xL Final solution volume 4 mL Sample weight 10 g... 

As can be derived that only ffl influences the broadening of a migrating zone, when the sample volume injected is very small, the detection path length is very narrow and the capillary diameter is very small. In such a case, is approximated by and Equation (33) can be simplified further to... 

When a particular component eluting at a certain retention volume is to be estimated, this approach can be outlined as follows. Since SEC is extremely reproducible, the peak shape, peak width and peak height are dependent on the amount of the species in the sample volume injected, sample volume and retention time. From these factors the SEC peaks can be simulated or elution pattern of any species within the separation range can be plotted as a function of mass vs. retention volume. The analysis data supplies the concentration of this particular species over two or more 0.5 ml intervals. A match-up computer program has to be developed so that it can pick up the peak shape and concentration based on 3 or 4 data points at known Intervals. 

Additionally, the combination of trace enrichment and microbore columns can effectively increase the maximum sample volume injectable without seriously degrading efficiency. Slais et al. (29) evaluated this combination for the determination of polynuclear hydrocarbons and chlorinated phenols in water. By using reversed-phase HPLC and am-perometric detection, Slais et al. (29) reported lower limits of detection from 20 to 280 ng/L of water (parts per trillion) when 1-mL sample enrichments were carried out directly on the analytical microbore column. 

If evaporation inside the syringe needle cannot be avoided, use 5- or 10-/d syringes, resulting in a minimum sample volume injected corresponding to the needle volume. Injection of 0.5-l- d volumes improves elution from the syringe needle. Splitless injection maximum sample volume around 2 pi (1 pi plus needle volume). 

The main problem interfacing a fraction collector with CE is the small sample volumes injected in CE. Many commercial instruments provide a means for sample collection. 

When transferring a method from a standard-bore column (4.6 mm I.D.) to a small-bore column, the sample volume injected is decreased according to the equation... 

Fig. 4.3 Chromatogram of an extract of river water measured with system A. Sample volume injected, lOOpL ...

In bioanalysis, extracted samples are usually stored in either autosampler vials or wells in a plate (such as 96-well plate) sealed with pierceable caps or covers. During injection, the autosampler needle has to pierce the caps or covers to load samples. The debris may completely or partially block the autosampler needle, which would result in no sample or variably low sample volumes injected. Accordingly, no or randomly low IS responses are observed. As most autosamplers have a built-in needle flushing mechanism, the debris in the needle might be flushed out later partially or completely. Therefore, the injected volume can be back to normal at a later time without an operator s intervention. Apparently, when a needle will be blocked and when the blocked needle will be cleared by flushing, as well as how it will be blocked (completely or partially) are difficult to predict. Hence, there would be no clear pattern for this type of IS variations. However, the affected injections normally have lowered IS responses (Fig. 9). Despite lowered IS responses, the accuracy of quantitation can usually be maintained except for situations where no or very low amount of samples are injected, resulting in responses outside the limit of linear range or unacceptable S/N. 

Split/splitless injector in the split mode only a portion of the injected sample (typically, 1 part in 50) reaches the column. The rest is vented to waste. A split injector is used for concentrated samples (> 0.1 mgmL for FID see p. 215). In the splitless mode all the sample volume injected passes through to the column. It is used, in this mode, for trace samples (< 0.1mgmL for FID). 

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. 

As the analytes to be assayed are nearly always present in minute amounts, preconcentration steps are almost always necessary. The techniques used for this purpose have been mostly adopted from analytical procedures using gas or liquid chromatography as the separation step [5]. This fact is emphasized here because the appropriate adjustment of the sample preparation is necessary because the original procedures do not respect the fact that the sample volume injected into a CE system represents a few nanoliters only and requires a relatively high concentration of analytes assayed. However, in selected types of analysis, direct sample application is also possible (for a review, see Ref. 3). 

Large sample volume. —> Inject small sample volumes. 

If all peaks are broadened, possible causes include a large sample volume injected, or a viscous mobile phase, or a column that has lost its efficiency, possibly due to the... 

Figure 1.7 HPLC chromatogram of organic acids analysis of Prosecco grape must sample. 1. tartaric acid, 2. malic acid, 3. citric acid, 4. shikimic acid. Analytical conditions Lichrospher 100 RP-18 (250 x4mm, 5p,m) column (Merck, Darmstadt, Germany) at room temperature, detection at wavelength 210 nm, sample volume injected 20/xL solvent H3P04 5 x 10 3 M with isocratic elution at flow rate 0.6 mL/min...

Figure 1.8 Chromatogram relative to HPLC analysis of sugars in a grape must sample. Analytical conditions column Aminex HPX-87H (300 x 7.8mm, 9p.m) (Bio-Rad Laboratories, Richmond, CA) at 60°C detector refractometer sample volume injected 20p,L solvent H2S04 0.013 N with isocratic elution at flow rate 0.6 mL/min...

H3P04 10 3 M and B) methanol and the elution gradient program reported in Table 2.8. Detection is performed at wavelength 320 nm (sample volume injected 25 xL). 

Figure 2.19 Chromatogram relative to analysis of monomer catechins in the skins extract (fraction diethyl ether from Ci8 cartridge) (sample volume injected lOp-L). 1. (+)-catechin, 2. (—)-epicatechin...

Injector Temperature 200°C, sample volume injected 0.5 pL, splitless injection... 

---