Extra-column volumes

Ve extra-column volume from valves, tubing, unions, etc. 

It must be pointed out that (Vm) refers to the volume of mobile phase in the column and not the total volume of mobile phase between the injection valve and the detector (Vo)- In practice, the dead volume (Vo) will include all the extra column volumes (Ve) involved in the sample valve, connecting tube detector cell etc. 

In some cases, (Ve) may be sufficiently small to be ignored, but for accurate measurements of retention volume, and particularly capacity ratios, the actual volume measured should always be corrected for the extra column volume of the system and equation (11) should be put in the form... 

In 2001, Valko et al. reduced the column length to only 50 mm and increased the flow rate to 2mLmin [42]. The gradient time was diminished to 2.5 min with a gradient cycle time of 5 min. Measurement of CHI and evaluation of log P were excellent with a 3-fold improved productivity. In these conditions, the system dwell volume (Vd) becomes essential and only dedicated chromatographic devices with Vjy lower than 0.8 mL can be used [42]. Special attention should be paid to the injected volume, which must remain lower than 3 pL to avoid any overloading or extra-column volume contributions. 

Several theoretical and empirical studies have been made of the impact of column ovens on separations [84]. The two most important factors that must be considered are the preheating of the mobile phase before it enters the column and the extra-column volume between the injector and the column. Work by Djordevic et al. [76] provides some of the most dramatic evidence for the impact of pre-heating the mobile phase. Their measurements of the axial temperature gradient in 4.6 mm columns at typical flow rates are shown in Table 9.2. 

There are a number of limitations on the use of extremes of temperature in HPLC. Clicq et al. [91] note that instrumental issues become increasingly limiting as one goes to very high temperatures and flow rates. They suggest that most separations will occur below 90°C where there are less instrumental constraints. As detailed below, column bleed can limit the selection of columns. Highspeed separations require a faster detector response than many systems allow and constrain extra column volume. This is especially true for narrow bore columns and sub-2 jam particles. In many cases, the additional speed gained above the temperature limits of commercial HPLC ovens will not be worth the additional expense and complexity required. For macromolecules, the effect of extreme pressure can also impact retention time as noted by Szabelski et al. [92]. 

The capacity ratio of a solute, (k ), was introduced to develop a chromatographic measurement, simple to calculate, independent of flow-rate and one that could be used in solute identification. Although helpful, the capacity ratio is so dependent on the accurate measurement of extra column volume and on very limited solute exclusion by the support and stationary phase, that it is less than ideal for solute identification. An alternative measurement, the separation ratio (a) was suggested where, for two solutes (A) and (B),... 

M mass of solute to be separated N number of effective theoretical plates P pressure Q flow rate R resolution S peak capacity Sm specific heat of mobile phase Ss specific heat of adsorbent Sg specific heat of detector cell walls V volume in conventional units Vo system dead volume Vr retention volume V r corrected retention volume Vm volume of mobile phase in the column Vs volume of stationary phase in the column Ve extra column volume... 

The volume of a chromatography system outside of the column from the point of injection to the point of detection is called the dead volume, or the extra-column volume. Excessive dead volume allows bands to broaden by diffusion or mixing. Use short, narrow tubing whenever possible, and be sure that connections are made with matched fittings to minimize dead volume and thereby minimize extra-column band spreading. 

Zero Dead Volume—Fittings designed to leave no extra column volumes that might cause band spreading or remixing of peaks. 

TABLE 17-5. Extra-column Volumes and Band-Spreading of Different Commercial... 

System Detector Cell Volume, Path Length Fec [rL] (Extra-column Volume) Wec [pL] (Extra-column Band Spreading)... 

Spreading for some commercial instruments. While instrument bandspreading is related to the extra-column volume, it does not correlate exactly due to the complexity of flow through tubing, connections, injection systems, and detector flow cells. 

After the injector, the smallest possible extra-column volume is required. This means that tubing with the narrowest possible i.d. should be used, in the shortest possible lengths. Commercial tubing is available with i.d. down to around 0.15 mm. 

The corrected retention volume, F, will be the difference between the retention volume and the dead volume Vq, which, in turn, will include the actual dead volume Vrn and the extra column volume F. Thus,... 

Fig. 1 Diagram depicting the retention volume, corrected retention volume, dead point, dead volume, and dead time of a chromatogram. Fq total volume passed through the column between the point of injection and the peak maximum of a completely unretained peak F total volume of mobile phase in the column F (a) retention volume of solute A F (a) corrected retention volume of solute A F extra column volume of mobile phase volume of mobile phase, per theoretical plate vy. volume of stationary phase per theoretical plate distribution coefficient of the solute between the two phases n number of theoretical plates in the column Q column flow rate measured at the exit.

The variance (the square of the standard deviation) of the observed peak can be expressed as the sum of the peak variances caused only by the contribution of the column (c7p) and all the contributions to the peak broadening due to the extra-column volume (Cex). This is expressed as... 

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