HPLC-FTIR

SDS-PAGE, MALDI TOP MS, 2-D Gel HPLC, GC, GC-MS GC, GC-MS HPLC, FTIR Diastase analysis GC, enzymatic assay LC, LC-MS, CE Pollen analysis AES and ICP-AES... 

FTIR instrumentation is mature. A typical routine mid-IR spectrometer has KBr optics, best resolution of around 1cm-1, and a room temperature DTGS detector. Noise levels below 0.1 % T peak-to-peak can be achieved in a few seconds. The sample compartment will accommodate a variety of sampling accessories such as those for ATR (attenuated total reflection) and diffuse reflection. At present, IR spectra can be obtained with fast and very fast FTIR interferometers with microscopes, in reflection and microreflection, in diffusion, at very low or very high temperatures, in dilute solutions, etc. Hyphenated IR techniques such as PyFTIR, TG-FTIR, GC-FTIR, HPLC-FTIR and SEC-FTIR (Chapter 7) can simplify many problems and streamline the selection process by doing multiple analyses with one sampling. Solvent absorbance limits flow-through IR spectroscopy cells so as to make them impractical for polymer analysis. Advanced FTIR... 

There is a need for increased chromatography-FTIR sensitivity to extend IR analysis to trace mixture components. GC-FTIR-MS was prospected as the method of choice for volatile complex mixture analysis [167]. HPLC-FT1R, SFC-FTIR and TLC-FTIR are not as sensitive as GC-FTIR, but are more appropriate for analyses involving nonvolatile mixture components. Although GC-FTIR is one of the most developed and practised techniques which combine chromatography (GC, SFC, HPLC, SEC, TLC) and FUR, it does not find wide use for polymer/additive analysis, in contrast to HPLC-FTIR. 

The flow-cell interface in HPLC-FTIR, first reported in 1975 [492], is the most straightforward. Flow-cells consist of two IR-transparent windows (KBr for... 

Microbore HPLC-FTIR detection limits are about 10 times lower than analytical-scale HPLC-FTIR detection limits. The lowest reported LC-FTIR detection limits are approximately 100-1000 times higher than the best GC-FTIR detection limits. The main characteristics of flow-cell HPLC-FTIR are summarised in Table 7.44. Because of mobile-phase interferences, flow-cell HPLC-FTIR is considered as a powerful tool only for the specific detection of major components but is otherwise a method of limited potential, and SFE-SFC-FTTR has been proposed as an alternative [391]. 

The obvious alternative for the in-line flow-through cell in HPLC-FTIR is mobile-phase elimination ( transport interfacing), first reported in 1977 [495], and now the usual way of carrying out LC-FTIR, in particular for the identification of (minor) constituents of complex mixtures. Various spray-type LC-FTIR interfaces have been developed, namely, thermospray (TSP) [496], particle-beam (PB) [497,498], electrospray (ESP) [499] and pneumatic nebulisers [486], as compared by Som-sen et al. [500]. The main advantage of the TSP-based... 

Table 7.44 Main characteristics of flow-cell HPLC-FTIR Advantages...

Better sensitivity (low-to-mid ng range) than flow-cell HPLC-FTIR... 

For microbore HPLC, with a flow of less than lOOpLmin-1, off-line LC-FT1R has been developed using matrix isolation techniques. The solutes are deposited on a moving IR salt window [504] or on a rotating plated disc [486], and are measured afterwards with the aid of a FITR microscope or a reflectance accessory. FTIR detection was first applied to the analysis of microbore HPLC eluent by Teramae and Tanaka [505]. In microbore HPLC-FTIR the amount of mobile phase required for separation is much less than for conventional scale HPLC. This simplifies both flow-cell and mobile-phase elimination interfaces. Flow-cell... 

Table 7.46 shows the LC-FTIR interface detection limits. Detection limits approaching those for GC-FHR light-pipe interfaces have been reported for flow-cell HPLC-FTIR when IR-transparent mobile phases are employed. For both the moving-belt and thermospray LC-MS couplings the detection limits are in the ng range. Selective evaporation consisting of fraction collection followed by DRIFT identification achieves a detection limit of 100 ng. 

Successful combination of a chromatographic procedure for separating and isolating additive components with an on-line method for obtaining the IR spectrum enables detailed compositional and structural information to be obtained in a relatively short time frame, as shown in the case of additives in PP [501], and of a plasticiser (DEHP) and an aromatic phenyl phosphate flame retardant in a PVC fabric [502], RPLC-TSP-FTIR with diffuse reflectance detection has been used for dye analysis [512], The HPLC-separated components were deposited as a series of concentrated spots on a moving tape. HPLC-TSP-FTIR has analysed polystyrene samples [513,514], The LC Transform has also been employed for the identification of a stain in carpet yarn [515] and a contaminant in a multiwire cable [516], HPLC-FTIR can be used to maintain consistency of raw materials or to characterise a performance difference. 

Norton KL, Lange AJ, Griffiths PR. A unified approach to the chromatography-FTIR interface GC-FTIR, SFC-FTIR and HPLC-FTIR with subnanogram detection limits. HRC-J. High Resolut. Chromatogr. 1991 14 225-229. 

The HPLC-FTIR technique has recently been used to identify six catechins and two methyl-xanthines present in green tea extracts." " A reversed-phase separation of the compounds was performed on a C-18 column equilibrated at 30°C using an isocratic mobile phase of acetonitrile-0.1% formic acid (15 85), prior to introduction to the deposition interface linked to the FTIR detector. The solvent was evaporated at 130°C and spectra were collected every 6 sec during the run. Two distinct designs for HPLC-FTIR interfaces have been developed flow cells and solvent elimination systems. Flow cell systems acquired spectra of the eluent in the solvent matrix through IR transparent, nonhydroscopic windows. The... 

For HPLC-FTIR, GPC-FTIR, or SFC-FTIR, the design of the interface is more challenging since the mobile phases used for these chromatographic systems normally have strong infrared absorbencies thus, it is important to remove the mobile phase prior to measuring the spectrum. For the interface between the two systems flow-cells or mobile-phase elimination techniques may be used. Some recent developments point toward the elimination of mobile-phase techniques. A microbore column can help to reduce the mobile-phase volume in the system. ... 

FIGURE 8 Example of HPLC-FTIR chromatograms for three drugs, acetaminophen, caffeine and dodecanolactam (top). Corresponding FTIR spectra are shown at the bottom (reproduced with permission from Bourne, I998). 

HPLC-FTIR has been successfully applied to the rather difficult identification of anabolic steroids (20) difficult, because steroids encompass a large number of natural and synthetic compounds with minor variations in molecular configuration. Despite the structural similarities of fluoxymesterone, testosterone, methyl testosterone, and epitestosterone, the distinct spectral signatures of these four compounds, which are all 17-hydroxysteriods with identical backbone structure, were recognizable. 

This methodology easily can be extended to the analysis of combinatorial chemistry samples. In combinatorial chemistry, the combination of chromatography and MS has proved a powerful method for substance identification based primarily on molecular weight. Analogously, HPLC-FTIR provides powerful identification possibilities, but based on molecular structure. As identifying a substance by MS requires knowledge about its possible molecular structure, the two techniques form a highly complementary system of identification. 

Since HPLC-FTIR is a high-throughput automatable hyphenated technique, it will be a powerful analytical tool in the burgeoning field of combinatorial chemistry. 

Providing identification based on molecular structure, HPLC-FTIR is therefore complementary to LC-MS. 

Coupling with spectroscopic techniques (HPLC-UV, HPLC-FTIR, HPLC-MS, HPLC-NMR), see Section 6.10. 

Figure 10.21 GramSchmidtprocess. Three-dimensional pre-recorded reconstruction between 1400 and 900 cm of a solution containing 10 mg/mL of three sugars, saccharose, glucose and fructose, by HPLC/FTIR (volume injected =50p,L) from KeUer R. etal. 1997 Anal Chem 69, 4288.

HPLC sample flow-cells have internal volumes of less than 20 pi, with a typical path length of 1mm and window area of 2-3 mm (Figure 7.13c). NaCl or KBr window materials may be used with many organic solvents and PTFE and polyethylene windows are available for both organic and aqueous based solvents. Narrow bore or microcolumns (2-3 mm i.d.) have lower flow volumes than normal columns, typically 0.3-0.5mlmin and are therefore ideally suited for infrared detectors. Micro-HPLC-FTIR techniques may employ a direct flow cell or solvent elimination techniques [16 18]. Considerable care is required to match the chromatographic system with the sample cell to avoid loss of resolution. 

Coupling of chromatography and spectroscopy ( chromatoscopy ) e.g. HPLC-MS(MS), HPLC-FTIR, HPLC-NMR, HPLC-NMR-MS. 

HPLC-FTIR suffers greatly from the inherent interference of the large excess of mobile-phase solvent. Since the common solvents are only simple organic mole-... 

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