Detectors, HPLC saturation

Coupled LC-LC can separate high-boiling petroleum residues into groups of saturates, olefins, aromatics and polar compounds. However, the lack of a suitable mass-sensitive, universal detector in LC makes quantitation difficult SFC-SFC is more suitable for this purpose. Applications of multidimensional HPLC in food analysis are dominated by off-line techniques. MDHPLC has been exploited in trace component analysis (e.g. vitamin assays), in which an adequate separation for quantitation cannot be achieved on a single column [972]. LC-LC-GC-FID was used for the selective isolation of some key components among the irradiation-induced olefinic degradation products in food, e.g. dienes and trienes [946],... 

Fig. 30 Silver ion high-performance liquid chromatography (Ag-HPLC-FID) with flame ionization detector (FID) analysis of the triacylglycerols of chromatographed Crepis alpina seed oil. Ag-HPLC-FID conditions 0.5-mg sample 5-micron Chromspher Lipids column (Chrompack International, Middelburg, The Netherlands) (4.6 X 250 mm) mobile phase 0.5% acetonitrile in hexane (v/v) flow rate 1.0 ml/min FID. Chromatogram peak triacylglycerol fatty acid abbreviations S, saturated (palmitic and stearic) O, oleic L, linoleic and Cr, crepenynoic fatty acids.

There are excellent HPLC systems available on the market today, yet there is one area of concern with this instrumentation, and this rests with the detection units. Certainly the most widely used detector system employs a low dead-volume micro-ultraviolet detector. This latter unit operates near 200 nm and detects mainly unsaturated linkages in phospholipids (or lipid) samples. Some contribution by carbonyl functions can be expected. This approach is an advantage when the sample under study contains olefinic groups, but will not detect those with saturated side (hydrocarbon) chains. An alternative detector is the refractive index monitor which is often called a universal detector, since it is based on the concept that the refractive index of the solvent changes when a solute is present. The drawback of the latter unit lies in its sensitivity, which is approximately 15- to 20-fold less than that of the ultraviolet monitor. 

Alternative detection methods such as mass spectrometry, ELS or nitrogen detector are in most cases inappropriate due to the octanol saturated aqueous media. An alternative is the use of standard HPLC reversed phase gradient systems (Valko). These systems do not represent true octanol water partitions, but are also good lipophilicity parameters by themselves. 

Figure 9.151 Determination of strictosidine synthetase activity by HPLC. Codeine (a), tryptamine (b), and strictosidine (c) were separated on a 4.0 (i.d.) X 250 mm LiChrosorb RP-8 Select B column at a flow rate of 1.0 mL/min. Incubation was for 30 minutes at 30°C with enzyme from Catharanthus roseus after ammonium sulfate precipitation (35-50% saturation) and gel filtration on Sephadex G-25, in the presence of 100 mM fi-D-gluconolactone. Injection volume was 8 pL and the UV detector was set at 0.02 AUFS. (From Pennings et al., 1989.)...

Typical Response Peaks. Figure 2 shows some typical response peaks for the HPLC separation of the saturates and aromatics in a typical vacuum gas oil. The curve in the upper portion of the figure is the response for the sample using the RI detector. After the saturates peak appeared, the backflush valve was switched and the attenuation changed as indicated in the figure. The peaks were very sharp and symmetrical and appeared in less than 10-min lapsed time from the point of injection. Base line drift was minimal and the areas of the response peaks were obtained with a ball and disc integrator on the strip chart recorder. 

Figure 2. Typical response peaks for HPLC separation of saturates and aromatics. Column, 1-ft /i-PorasU soU vent, n-heptane (1 mL/min) sample, 10 fiL of vacuum gas oil in n-heptane detectors, refractive index, and Pye Unicam moving wire.

If necessary, a classical detector can be attached to the column outlet, preceeding the fraction collector. UV detectors are the easiest to install and use. A variable-wavelength detector allows selection of the monitoring wavelength. Such detectors should be equipped with the appropriate cell. Analytical cells, used on HPLC systems, have path lengths of 6-10 mm and are not suitable for use with preparative open columns, as the detector output will quickly become saturated. A more generally useful cell for open column detectors would be a cell with a path length of less than 0.5 mm. 

The main limitation to nebulizer HPLC-MS interfaces is that many common HPLC solvent modifiers such as H3PO4 are not tolerated, as they are not volatile and will saturate the mass detector, causing unacceptable losses in sensitivity. This problem can be circumvented using a belt-drive-interface system, but this interface presents other more mechanical problems. 

Under a nitrogen atmosphere, a hexane solution of butyllithium (650 pL, 1.00 nunol) was added to bromobenzene (157 mg, 1.00 mmol) in Et20 (0.5 itiL) at 0 X. The mixture was stirred at room temperature for 1 h and then cooled to -78 °C. Trimethoxyborane (104 mg, 1.00 mmol) was added to the reaction mixture. The mixture was stirred at -78 C for 30 min and then at room temperature for 1 h. To the mixture were added H2O (18 mg, 1.00 mmol), isopropyl tra s-2-hexenoate (262) (62 mg, 0.40 mmol), and a solution of Rh(acacXC2H4)2 (3.1 mg, 12 pmol) and (S)-BINAP (9.0 mg, 14 pmol) in dioxane (2.0 mL). The whole mixture was heated at 100 C for 3 h. Addition of saturated aqueous sodium bicarbonate followed by ethyl acetate extraction and chromatography on silica gel (hexane ethyl acetate = 10 1) gave 90 mg (96% yield) of isopropyl 3-phenylhexanoate 264 as a colorless oil. HPLC analysis was performed on a Shimadzu LC-9A (Shimadzu Corp. Nakagyo-ku, Kyoto, Japan) and a JASCO PU- 980, with a JASCO UV-970 UV detector (Jasco Inc., Easton, MD, U.S.A), liquid chromatographic system with chiral stationary phase columns Chiralcel OD-H, OJ and OG, (95% ee). ... 

As shown in Figure 3.8, an increased injection volume can have a negative effect on the column efficiency. Therefore, the injection volume should be appropriately scaled down when an HPLC method is transferred to a UHPLC method or vice versa to achieve the same sensitivity and avoid overloading or detector saturation and extracolumn band-broadening. The scaling factor is mainly based on column dimension, as shown in Eq. (3.26). However, lower injection volumes than calculated can often be used on UHPLC to achieve the same sensitivity due to enhanced peak heights from use of the high-resolution columns and low carryover from the injector ... 

Experiments can be performed in a liquid chromatograph (HPLC), complete with pump, injector, and detector. The catalyst sample, in a sample holder surrounded by a thermostatted jacket, is placed in place of the chromatographic column and the chosen liquid is continuously eluted on the solid. In the adsorption test, pulses of a given amine solution of known concentration are sent at fixed time intervals onto the catalyst sample at a constant liquid flow rate. The non-adsorbed amounts of amine after each pulse are detected and measured up to sample saturation. The amount of amine adsorbed (mequiv/g) on the sample after the i-th pulse is calculated by the following equation ... 

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