Chromatogram milk proteins

Determinative and confirmatory methods of analysis for PIR residue in bovine milk and liver have been developed, based on HPLC-TS-MS (209). Milk sample preparation consisted of precipitating the milk proteins with acidified MeCN followed by partitioning with a mixture of -butylchloride and hexane, LLE of PIR from aqueous phase into methylene chloride, and SPE cleanup. The dry residue after methylene chloride extraction was dissolved in ammonium hydroxide, and this basic solution was transferred to the top of Cl8 SPE column. The PIR elution was accomplished with TEA in MeOH. For liver, the samples were extracted with trifluoroacetic acid (TFA) in MeCN. The aqueous component was released from the organic solvent with n-butyl chloride. The aqueous solution was reduced in volume by evaporation, basified with ammonium hydroxide, and then extracted with methylene chloride. The organic solvent was evaporated to dryness, and the residue was dissolved in ammonium acetate. The overall recovery of PIR in milk was 94.5%, RSD of 8.7%, for liver 97.6%, RSD of 5.1 %. A chromatographically resolved stereoisomer of PIR with TS-MS response characteristics identical to PIR was used as an internal standard for the quantitative analysis of the ratio of peak areas of PIR and internal standard in the pro-tonated molecular-ion chromatogram at m/z 411.2. The mass spectrometer was set for an 8 min SIM-MS acquisition. Six samples can be processed and analyzed in approximately 3 hours. 

Figure 15.14 presents a separation of milk proteins. Draw a calibration curve which corresponds to this chromatogram. 

Figure3.18 Separation of the milk proteins buffer. In both chromatograms, the same P-globulin B (pi 5.2) and p-lactoglobulin A amount of proteins was injected, but in 0.1 and...

Many proteins and polymers have been analyzed on SynChropak GPC and CATSEC columns. Table 10.6 lists some of the published applications. The use of a surfactant to analyze the caseins in milk is illustrated in Eig. 10.12. Viruses have also been analyzed on SynChropak GPC columns, as seen in the chromatogram from Dr. Jerson Silva of the University of Illinois (Pig. 10.13). Dr. Nagy and Mr. Terwilliger analyzed cationic polymers on a series of CATSEC columns using differential viscometry as detection (Pig. 10.14) (9). 

Only a few works report a simple combination of conventional sample cleanup procedures with UV detection. Assay of CLO (OXA was internal standard) based on a protein precipitation using MeCN in acidic solution (pH 3.6) and LLE with chloroform was reported for milk samples. The organic layer was evaporated to dryness, and the residue was reconstituted in the mobile phase and injected into the chromatographic system with UV detection. Relatively clean chromatograms and high recoveries (75-84%) were obtained using this assay (55). 

Fig. 150. Thin-layer chromatogram of phospholipids and other polar lipids of bovine milk [159 a]. 1 carbohydrate (lactose) and protein, 2 sphingomyelin, 3 phosphatidyl choline, 4 phosphatidyl serine, 5 phosphatidyl inositol, 6 phosphatidyl ethanolamine, 7 cerebroside dihexoside ( ), 8 cerebroside mono-hexoside ( ), P fatty acids, 10 neutral lipids. Adsorbent Silica gel HR. Solvents I, chloroform-methanol-water-28% aqu. ammonia (130 + 70+8 + 0.5) II, chloroform-acetone-methanol-acetic acid-water (50 + 20 + 10 + 10 + 5). Time 40 min in each direction. Indicator charring with chromic sulphuric acid solution...

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