Column, capillary overload

Numerous types of GC injectors have been manufactured over the past four decades. The most commonly used injection techniques have been reviewed and described by Grob, who correctly states that analysts must fully understand the techniques before they can make the most appropriate choice for their particular application(s). For most GC capillary column applications, the split/splitless, programmed-temperature vaporization (PTV) and on-column injectors remain the most popular. However, over the last few years, technology has progressed rapidly to provide injectors that allow more of the sample extract on to the GC column without overloading it. 

Injectors Sample injection devices range from simple syringes to fully programmable automatic injectors. The amount of sample that can be injected into a capillary column without overloading is small compared with the amount that can be injected into a packed column, and may be less than the smallest amount that can be manipulated satisfactorily by syringe. Capillary columns are therefore used with injectors able to split samples into two fractions, a small one that enters the column and a large one that goes to waste (split injector). Such injectors may also be used in a splitless mode for analyses of trace or minor components. 

When sample concentrations are much lower (ppm to ppb, or lower) one will wish for all the analytes to be transferred to the capillary column, and overloading will be less of a... 

The UV-Visible detector is the universal detector used in analytical and preparative CCC. It does not destroy solutes. It is used to detect organic molecules with a chromophore moiety or mineral species after formation of a complex (for instance, the rare earth elements with Arsenazo III ). Several problems can occur in direct UV detection, as has already been described by Oka and Ito 1) carryover of the stationary phase due to improper choice of operating conditions, with appearance of stationary phase droplets in the effluent of the column 2) overloading of the sample, vibrations, or fluctuations of the revolution speed 3) turbidity of the mobile phase due to difference in temperature between the column and the detection cell or 4) gas bubbling after reduction of effluent pressure. Some of these problems can be solved by optimization of the operating conditions, better control of the temperature of the mobile phase, and addition of some length of capillary tubing or a narrow-bore tube at the outlet of the column before the detector to stabilize the effluent flow and to prevent bubble formation. The problem of stationary phase carryover (especially encountered with hydrodynamic mode CCC devices) can be solved by the addition between the column outlet and UV detector of a solvent that is miscible with both stationary and mobile phases and that allows one to obtain a monophasic liquid in the cell of the detector (a common example is isopropanol). 

Because of the length and small diameter of capillary columns, there is increased interaction between the compounds in the mixture and the stationary phase. Capillary columns are, therefore, much more efficient in separating compounds with similar properties than with packed coliunns, but only if a dilute solution of a mixture of compounds is injected into the column. In order to obtain a satisfactory separation, you must dissolve about 1 drop of a mixture in about 2 mL of a solvent such as methylene chloride or pentane. About 1 fiL of this dilute sample is injected onto the coliunn. In contrast, 1 to 10 fiL of an undiluted sample can often be analyzed on a packed column without overloading the coliunn. These expensive capillary columns must be purchased from commercial companies specializing in their manufacture. Sensible researchers would never try to make their own capillary columns ... 

Injection systems of a capillary gas chromatography should fulfil two essential requirements (i) the injected amount should not overload the column (ii) the width of the injected sample plug should be small compared with band broadening due to the chromatographic separation. Good injection techniques are those which achieve optimum separation efficiency of the column, allow accurate... 

The parameters that influence the injection plug width will be discussed in a further section of this chapter. In practice, it is necessary to keep the width of the injection plug smaller than 1% of the total length of the capillary, in order to avoid column overloading [27]. 

As implied above, the appropriate range of sample injection volume depends on column diameter. As we will see in the next section, column diameters vary from capillary size (0.2 to 0.3 mm) to i/8 and i/4 in. Table 12.2 gives the typical injection volumes suggested for these column diameters. The capillary columns are those in which the overloading problem mentioned above is most relevant. Injectors preceding the 1/8 in. or larger columns are not split. 

The packed column can be from 2 to 20 ft in length, typically has a diameter of V8 or V4 in., and has small particles, often coated with a thin layer of liquid stationary phase, packed in the tube. The open-tubular capillary column can be up to 300 ft in length, has an extraordinarily small diameter (capillary), and has the liquid stationary adsorbed on the inside surface of the tube. In terms of separation ability, the open-tubular capillary column is better because the mixture components contact more stationary phase (column is longer) while passing through the column. The amount injected for the open-tubular capillary column must be much less (0.1 mL maximum, as opposed to 20 mL for the V8-in. packed column) because the column diameter is much less and a greater volume would overload it. 

In direct injection, all injected material is carried onto the column by the mobile phase. This eliminates the possibility of sample discrimination in the inlet, but can overload capillary columns and so is more commonly used with packed columns and wide-bore capillary columns. 

One method that prevents overloading of narrow-bore capillary columns is split injection. The inlets are usually bimodal split/splitless inlets, and either mode can be selected for a given analytical method. In the split mode, the sample is rapidly vaporized in the inlet and a portion is introduced into the column in a narrow band with carrier gas, while the rest of the sample is vented (Dybowski and Kaiser, 2002). The amount introduced can vary for each method and is chosen as a ratio, e.g., 1 100. Easily vaporized compounds may preferentially vent, leading to the introduction of a nonrepresentative sample to the column (Watson, 1999). When the splitless mode is chosen, the entire sample is introduced into the column and the vent is opened after a predetermined period of time, to flush the excess solvent from the injector (Dybowski and Kaiser, 2002). 

Capillary columns are easily overloaded by the level of excretion detectable in acutely ill patients. Any profile with the characteristic appearance of oversaturated peaks (see Fig. 3.1.5a and b) should be repeated after proper dilution when quantitation has to be provided using a GC-MS TIC method (Fig. 3.1.6). 

There are three injection techniques for introducing a sample into a GC equipped with a capillary column split injection, splitless injection, and on-column injection. Split injection is the most often used injection technique. When a certain amount of FAME sample (1 to 3 ll) is introduced into the GC injector that is normally set at a temperature much higher than the boiling point of the solvent, the solvent vaporizes instantly in the carrier gas and creates a large volume of gas that contains all of the injected FAME in it. The carrier gas that contains the FAME is then divided into two streams from the injector one is directed onto the column, and the second is vented to the atmosphere, clearing the sample out of the injection chamber momentarily. This way, only a limited amount of sample is introduced into the column, to avoid column overloading, and injection time is short, to avoid peak broadening. 

Two types of injectors are frequently employed. For packed column SFC, a standard six port rotary valve with an external sample loop of 1-10 pL has proven to be quite reliable. For capillary column SFC, a similar rotary valve with an internal "loop of 0.2 to 0.5 pL is typically employed. Frequently the rotor is pneumatically actuated in a very rapid fashion to allow only a small fraction of sample to be introduced ("time-split ) this is done to avoid column overload. Alternatively, the flow from the injector is split off in the same fashion as in GC. A disadvantage of the latter mode is the potential for sample discrimination. 

Figure 5 compares chromatograms of A8- and A9-THC-TMS and 11-hydroxy-A8-THC-TMS obtained on a glass capillary column and a 6ft. x 2mm I.D. packed column. The retention times of the cannabinoids are comparable, but the resolution and sensitivity achieved on the capillary column is far superior. The capacity of the capillary column is of course far less than that of the packed column. Nevertheless, by using a Grob-type splitless injector (7) we are able to inject several microliters of solution containing up to 50 ng of each cannabinoid without overloading the column. Use of a support-coated open tubular column should increase the column capacity, but at some sacrifice of resolution and sensitivity. 

The main advantage of OT CEC is that separation efficiency can be doubled using this type of column. The trade-off is that the OT columns can easily be overloaded and therefore require a sensitive detection system. The small diameter of these columns precludes the use of UV detection, and fluorometric detection or mass spectrometry (MS) needs to be used. The use of fused silica capillaries with a bubble cell at the detection window has been reported as an alternative to employ UV detection. This features limit, to a certain extent, the range of practical applications of OT CEC. 

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