Phase separation particle size analysis

The most useful parameters commonly measured to assess the effect of stress conditions on emulsions include phase separation, rheological property determination, electrical property measurements, and particle size analysis. 

In terms of particle size and column dimensions, stationary phases with particle sizes of 5-10 /rm in 250 x 4.6-mm analytical columns are most common. HPLC columns with smaller particle sizes (2-3 /rm) have also been prepared and used. Such columns have the advantages of higher efficiency, better resolution, and shorter analysis times. For example, the triglycerides of cocoa butter were baseline separated with columns that contained 2-3-/tm particles. Research and development of columns with smaller particles is very active. 

It is seen that, although the dimensions and particle sizes may not be precisely matched, all three columns are of a size closely similar to those commercially available with, perhaps, the exception of the long high efficiency column. The small 3 cm column is excellent for the preliminary assessment of a sample. As a result of its size it does not use large volumes of solvent and can be quickly reconditioned after a separation in readiness for the next run. It is very convenient for choosing the best phase system in method development. The other columns would be chosen on a basis of the efficiency required to separate the critical pair in the reduced chromatogram of the sample for analysis. 

The column used for blood serum analysis was 100 cm long, 1 mm in diameter and packed with RP 18 reversed phase having a particle size of 10 pm. A concave gradient program was used to develop the separation over a period of 45 min. at a flow rate of 50 pl/min. The initial solvent was 75% methanol 25% water and the final solvent was pure methanol. 

These columns have been used for separation of proteins of over 200 kDa MW in our experiments as shown by analysis using a ID gel. In addition, columns with larger particle sizes have been used to separate proteins of over 400 kDa (55-56). The NPS RP-HPLC method provides a liquid phase method for separating large intact proteins for further analysis. More specifically, it provides a means of separating proteins for interfacing to mass spectrometric analysis. 

It was stated that the chromatographic analysis of the flavonoid profde may help the determination of taxonomic relationships between these species [125], The optimization of the separation of flavonoid glycosides of Mentha piperita (Laminaceae) was carried out on silica, amino, cyano and C18 HPTLC statinoary phases. The investigation was motivated by the spasmolytic, carminative and cholagogue characteristics of the plant. Air-dried and powdered leaves of peppermint (300 g) were extracted with methanol-water 1 1 v/v at ambient temperature. The suspension was filtered, concentrated to 200 ml acidified to pH 3 with formic acid and separated in an ODS column (400 X 40 mm i.d. particle size 40 pm). 

RP-HPLC has also been used for the analysis of flavan-3-ols and theaflavins during the study of the oxidation of flavan-3-ols in an immobilized enzyme system. Powdered tea leaves (20Qmg) were extracted with 3 X 5 ml of 70 per cent aqueous methanol at 70°C for lQmin. The combined supernatants were filtered and used for HPLC analysis. Flavan-3-ols were separated in a phenyl hexyl column (250 X 4.6 mm i.d. particle size 5 /im) at 30°C. Solvents A and B were 2 per cent acetic acid in ACN and 2 per cent acetic acid in water, respectively. Gradient elution was 0-lQmin, 95 per cent B 10-4Qmin, to 82 per cent B to 40-5Qmin 82 per cent B. The flow rate was 1 ml/min. Theaflavins were determined in an ODS column (100 X 4.6 mm i.d. particle size 3pm) at 30°C. The flow rate was 1.8 ml/min and solvent B was the isocratic mobile phase. The data demonstrated that flavan-3-ols disappear during the oxidation process while the amount of theaflavins with different chemical structures increases [177],... 

Normal-phase HPLC has also found application in the analysis of pigments in marine sediments and water-column particulate matter. Sediments were extracted twice with methanol and twice with dichloromethane. The combined extracts were washed with water, concentrated under vacuum and redissolved in acetone. Nomal-phase separation was performed with gradient elution solvents A and B being hexane-N,N-disopropylethylamine (99.5 0.5, v/v) and hexane-2-propanol (60 40, v/v), respectively. Gradient conditions were 100 per cent A, in 0 min 50 per cent A, in 10 min 0 per cent A in 15 min isocratic, 20 min. Preparative RP-HPLC was carried out in an ODS column (100 X 4.6 mm i.d. particle size 3 jum). Solvent A was methanol-aqueous 0.5 N ammonium acetate (75 25, v/v), solvent B methanol-acetone (20 80, v/v). The gradient was as follows 0 min, 60 per cent A 40 per cent A over 2 min 0 per cent A over 28 min isocratic, 30 min. The same column and mobile phase components were applied for the analytical separation of solutes. The chemical structure and retention time of the major pigments are compiled in Table 2.96. 

Fig. 3.38.The IUPAC names of Sudan azo dyes are as follows Sudan 1 = 1— [(2,4-dimethylphenyl)azo]-2-naphtalenol Sudan II = l-(phenylazo)-2-naphtol Sudan III = l-(4-phenylazophenylazo)-2-naphtol Sudan IV = o-tolyazo-o-tolyazo-beta-naphtol and Disperse Orange 13 = 4-[4-(phenylazo)-l-naphtylazo]-phenol. Azo dyes were separated in an ODS column (250 x 2.1 mm i.d. particle size 5 /xm) at 35°C. The isocratic mobile phase consisted of 0.1 per cent formic acid in methanol-0.1 per cent formic acid in water (97 3, v/v). The flow rate was 200 /xl/min. MS conditions were nebulizing and desolvation gas were nitrogen at the flow rates of 50 and 5551/h, respectively electrospray voltage, 3.0 kV cone voltage 25 V source temperature, 110°C desolvation temperature, 110°C. Azo dyes were extracted from the samples by homogenizing 1 g of sample with 10 ml of acetone, then the suspension was centrifuged and an aliquot of 3 ml of supernatant was mixed with 1 ml of deionized water, filtered and used for analysis. LC-ESI-MS/Ms SRM traces of standards and spiked samples are listed in Fig. 3.39. It was found that the detection and quantitation limits depended on both the chemical structure of the dye and the character of the accompanying matrix. LOD and LOQ values in chilli tomato sauce...

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