High performance liquid chromatography conditions optimization

JA Bietz, T Burnouf, LA Cobb, JS Wall. Gliadin analysis by reversed-phase high-performance liquid chromatography optimization of extraction conditions. Cereal Chem 61 124-129, 1984. 

High performance liquid chromatography techniques may be successfiilly applied to analyze phthalate esters. A 15 or 25 cm column filled with 5 or 10 pm silica-based packings is suitable. Short columns (3.3 cm x 4.6 mm), commonly called 3x3 columns, offer sufficient efficiency and reduce analysis time and solvent consumption Phthalate esters resolve rapidly on a 3 x 3 Supelcosil LC-8 column (3 pm packing) at 35°C and detected by a UV detector at 254 nm. Acetonitrile-water is used as mobile phase (flow rate 2 ml/min injection volume 1 mL). Other equivalent columns under optimized conditions may be used. 

FIGURE 6-23. High performance liquid chromatography analysis of fraction 2. This chromatogram shows that cortisone is eluted quantitatively from the SPE cartridge with 2 mL of 100% methanol. Now conditions can be optimized for a faster HPLC analysis. 

It was established that the reaction between 45 and 3,4,5-trimethoxybenzaldehyde 46 required cooling in an ice bath in order to obtain reproducible results and a cleaner product, and hence, microwave irradiation was not utilized in this step. Intermediate 47, thus obtained using conventional methods, was used for the synthesis of 43 under microwave conditions without further purification. Reaction of 47 with a large excess of guanidine under microwave irradiation for 30 min at 140°C afforded trimethoprim 43 in 40% yield, assessed by high-performance liquid chromatography (HPLC). Further optimization of these reaction conditions did not increase the yield of the product. 

Typical chromatograms obtained by high-performance liquid chromatography (HPLC) of the retinyl esters from human RPE are illustrated in Fig. 5. When conditions are optimized for isomer separation (normal phase, uj r half of Fig. 5), there are two major peaks (2, 4), each with a shoulder on the leading edge. Peak 2 is 11-cw-retinyl palmitate the shoulder corresponds to 11-cij-retinyl stearate. Peak 4 is all-trans-retinyl palmitate the shoulder corresponds to all-trans retinyl stearate. Peak 5 is all-rra/u-retinyl stearate, and peak 3 (which is sometimes composite) is unidentified at present. Peaks 2 and 4 were collected separately and injected onto a reverse-phase column. Under these conditions the two component esters were completely resolved, as shown in the lower half of Fig. 5 (peaks 2.1, 2.2 and 4.1, 4.2). 

Ozaki A, Shibasaki T, Mori H (1995) Specific proline and hydroxyproline detection method by post-column deri tization for high-performance liquid chromatography. Biosci Biotechnol Biochem 59 1764—1765 Sano K, Mitsugi K (1978) En rmatic production of L-cysteine from DL-2-amino-A2-thiazoline-4-carboxyiic acid by Pseudomonas thiazolinohilum optimal conditions for the enzyme formation and enzymatic reaction. Agric Biol Chem 42 2315-2321... 

Important areas of data processing include the use of chemometrics (Topic B5) to simplify complex data for characterizing materials, quantitative spectro-metric analysis using multiple wavelengths, and routines to optimize experimental conditions for high-performance liquid chromatography. 

Yodoshi, M. Tani, A. Ohta, Y Suzuki, S. Optimized conditions for high-performance liquid chromatography analysis of oligosaccharides using 7-amino-4-methylcoumarin as a reductive amination reagent. J. Chromatogr., A 2008, 1203, 137-145. 

Reaction conditions employed included the reaction in the presence or absence of external bases for the bisnucleophUes different concentrations of mononucleophiles and nucleophilic zwitterions. After a quick aqueous workup, a liquid chromatography coupled to mass spectrometry (LC-MS) analysis ofthe crude reaction mixture was performed. Reactions with high conversions (consumption of 27) and relatively cleaner product profiles were then separately optimized (when required) for better results. The reaction screening revealed that 27-ketoesters were better... 

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