Solute peak

Besides the solute peak, Figure 12.7 also shows a small peak eluted soon after the sample is injected into the mobile phase. This peak results from solutes that move through the column at the same rate as the mobile phase. Since these solutes do not interact with the stationary phase, they are considered nonretained. The time or volume of mobile phase required to elute nonretained components is called the column s void time, or void volume. 

From equation 12.1 it is clear that resolution may be improved either by increasing Afr or by decreasing wa or w-q (Figure 12.9). We can increase Afr by enhancing the interaction of the solutes with the column or by increasing the column s selectivity for one of the solutes. Peak width is a kinetic effect associated with the solute s movement within and between the mobile phase and stationary phase. The effect is governed by several factors that are collectively called column efficiency. Each of these factors is considered in more detail in the following sections. 

Using equation (10), the efficiency of any solute peak can be calculated for any column from measurements taken directly from the chromatogram (or, if a computer system is used, from the respective retention times stored on disk). The computer will need to have special software available to identify the peak width and calculate the column efficiency and this software will be in addition to that used for quantitative measurements. Most contemporary computer data acquisition and processing systems contain such software in addition to other chromatography programs. The measurement of column efficiency is a common method for monitoring the quality of the column during use. 

The solvent used was 5 %v/v ethyl acetate in n-hexane at a flow rate of 0.5 ml/min. Each solute was dissolved in the mobile phase at a concentration appropriate to its extinction coefficient. Each determination was carried out in triplicate and, if any individual measurement differed by more than 3% from either or both replicates, then further replicate samples were injected. All peaks were symmetrical (i.e., the asymmetry ratio was less than 1.1). The efficiency of each solute peak was taken as four times the square of the ratio of the retention time in seconds to the peak width in seconds measured at 0.6065 of the peak height. The diffusivities obtained for 69 different solutes are included with other physical and chromatographic properties in table 1. The diffusivity values are included here as they can be useful in many theoretical studies and there is a dearth of such data available in the literature (particularly for the type of solutes and solvents commonly used in LC separations). 

Equation (20) allows the efficiency of any solute peak, from any column, to be calculated from measurements taken directly from the chromatogram. Many peaks, if measured manually, will be only a few millimeters wide and, as the calculation of the column efficiency requires the width to be squared, the distance (x) must be determined very accurately. The width should be measured with a comparitor reading to an accuracy of 0.1 mm. 

Special solvents that are not components of the mobile phase, but are included in the sample to improve component solubility, will act as though they were solutes themselves. Each will produce a spurious peak somewhere on the chromatogram that must not be misinterpreted as a solute peak. Irrespective of the sample solvent, the solutes of interest must always be sufficiently soluble in the mobile phase to permit effective chromatographic development. 

In practice, it is probable that both of the effects discussed contribute to the overall peak asymmetry. Unfortunately, peak asymmetry varies in extent from the very obvious to the barely noticeable and because of this, peak asymmetry is often dismissed as the normal shape of a single solute peak. Such an assumption can cause serious errors in both qualitative and quantitative analysis. 

It is seen that the profile of the combined peaks is perfectly symmetrical and displays no hint that there are two solutes present. Obviously an absorption ratio curve from a diode array detector would quickly disclose the presence of the two components, as would an appropriate changes in mobile phase composition. However, there would be a further clue for the analyst to follow that would give warning of the "duplicity" of the peak. The double peak would be very broad and be inconsistent with the change in peak width of the other solute peaks with retention time. The peak width of a solute increases regularly with retention time but, unfortunately, the relationship is not smooth. There are good reasons for this, but they... 

Suppose that we now use a column in the system, and that the width of our solute peak is wy. This will be larger than w>b, because of the extra-column dispersion. The relation between the three peak widths is ... 

The absorbance ratio AnlAxi for the solute peak should be close to zero. If it is not, then this suggests that the peak is not what we think it is. For example, there may be another component that elutes at the same time, so the ratio method is a simple way of indicating the purity of the peaks. 

Figure 5.12 GALDI mass spectrum of shellac (from methanol solution). Peaks at m/z—570 are related to esters of aliphatic hydroxy acids with sesquiterpenoid carboxylic acids (see text and Table 5.4). Signals marked with crosses are contaminants in the spectrometer that accumulated over time (m/z 413, 469, and 507) peaks marked ( ) are contaminating graphite clusters from the matrix (m/z 264, 276, 288).

Figure 6. Comparison of experimental (—) and theoretical (—) absorption spectra for the yellow solution. Peaks in the theoretical curve are due to contributionsfromchains of length I and are labeled with the appropriate 1. (Reproduced with permission from Ref. 21. Copyright 1982, American Institute of Physics.)...

Chewable tablets and oral solution Peak plasma concentrations are achieved at approximately 1 to 2 hours. 

PAR is the peak area ratio of the solute peak area compared to the area of the internal standard peak. 

Flurbiprofen is given orally or rectally (150-200 mg/day, max. 300 mg/day) and as ophthalmic solutions. Peak plasma concentration appears 1 to 2 hours after oral administration. The plasma protein binding is about 99.5% and the elimination half-life is in the range of 3-5 h. 

A retention gap is used to improve peak shapes under certain conditions. If you introduce a large volume of sample (>2 pL) by splitless or on-column injection (described in the next section), microdroplets of liquid solvent can persist inside the column for the first few meters. Solutes dissolved in the droplets are carried along with them and give rise to a series of ragged bands. The retention gap allows solvent to evaporate prior to entering the chromatography column. Use at least 1 m of retention gap per microliter of solvent. Even small volumes of solvent that have a very different polarity from the stationary phase can cause irregular solute peak shapes. The retention gap helps separate solvent from solute to improve peak shapes. 

Experiment C is the same as B. but the split vent was opened after 30 s to rapidly purge all vapors from the injection liner. The bands in chromatogram C would be similar to those in B, but the bands are truncated after 30 s. Experiment D was the same as C, except that the column was initially cooled to 25 °C to trap solvent and solutes at the beginning of the column. This is the correct condition for splitless injection. Solute peaks are sharp because the solutes were applied to the column in a narrow band of trapped solvent. Detector response in D is different from A-C. Actual peak areas in D are greater than those in A because most of the sample is applied to the column in D. but only a small fraction is applied in A. To make experiment D a proper splitless injection, the sample would need to be much more dilute. 

The theoretical plate concept in chromatography is a popular approach to determining column efficiency (relative band broadening in the column). The number of theoretical plates, N, is related to the retention time and to the width of the solute peak by... 

Fig.4.26. Chromatogram of an ampoule solution. Peaks 1 = hyoscyamine 2 = scopolamine.

Quantification by external standard is the most straightforward approach because the peak response of the reference standard is compared to the peak response of the sample. The standard solution concentration should be close to that expected in the sample solution. Peak responses are measured as either peak height or area [41 ]. 

Sample introduction is a major hardware problem for SFC. The sample solvent composition and the injection pressure and temperature can all affect sample introduction. The high solute diffusion and lower viscosity which favor supercritical fluids over liquid mobile phases can cause problems in injection. Back-diffusion can occur, causing broad solvent peaks and poor solute peak shape. There can also be a complex phase behavior as well as a solubility phenomenon taking place due to the fact that one may have combinations of supercritical fluid (neat or mixed with sample solvent), a subcritical liquified gas, sample solvents, and solute present simultaneously in the injector and column head [2]. All of these can contribute individually to reproducibility problems in SFC. Both dynamic and timed split modes are used for sample introduction in capillary SFC. Dynamic split injectors have a microvalve and splitter assembly. The amount of injection is based on the size of a fused silica restrictor. In the timed split mode, the SFC column is directly connected to the injection valve. Highspeed pneumatics and electronics are used along with a standard injection valve and actuator. Rapid actuation of the valve from the load to the inject position and back occurs in milliseconds. In this mode, one can program the time of injection on a computer and thus control the amount of injection. In packed-column SFC, an injector similar to HPLC is used and whole loop is injected on the column. The valve is switched either manually or automatically through a remote injector port. The injection is done under pressure. 

In this section we will discuss some aspects of the more common type of solvent peaks, i.e. large signals which appear early in the chromatogram. Figure 4.14 shows a typical chromatogram in which three solute peaks are preceded by a large solvent peak. 

A given solute peak migrates at one-fourth the velocity of the mobile phase, R = 1/4. What is the value of the capacity factor k l... 

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