Column preparation purification

Another application shows the preparative purification and polishing of a therapeutic fusion protein with a humanized recombinant IgG protein. The fusion protein was expressed by the fermentation of baby hamster kidney cells. The filtered culture supernatant (155 liters) contained 2.2 g of IgG and 75.5 g of total protein. After the immunoglobulins were isolated by expanded bed adsorption and rebuffering, the IgG fraction was bound to Fractogel EMD SOj (M). This column achieved baseline separation of complete antibodies (fusion protein) from small amounts of antibodies lacking the fusion part. The resulting highly purified IgG fraction (110 ml) was diluted to 150 ml and... 

Cyclic peptide from 11 amino acids. Preparation by fermentation of Tolypocladium inflatum Gams with addition of DL-a-aminobutyric acid to the fermentation medium. Isolation by homogenization of mycelium, extraction with 90 % methanol and column chromatographic purification. 

Starting with 3 GBq [nC]carbon dioxide produced in a 14N(p,a)11C nuclear reaction, the radiochemical yield of 165 was 0.5 GBq at the end of preparative purification performed in Sep-Pak C18 columns. The methyl esters of prostaglandins have a high affinity for the specific binding sites141. 

Delincee and Radola100 used a commercial preparation, as well as fresh tomatoes, for the preparation, purification, and characterization of tomato pectinesterase. The tomatoes were pressed and then homogenized directly with ammonium sulfate at 70% saturation. The precipitate obtained was extracted with 0.3 M phosphate and repeatedly salted out with ammonium sulfate, and the product was separated on a column of Sephadex G-75. The pattern of separation was similar to that in preceding work.50,97 A detailed study of the size properties of pectinesterase was conducted by gel-filtration and sedimentation analysis.100 By column and thin-layer gel-filtration on Sephadex G-75, the approximate molecular weight of a number of preparations of tomato pectinesterase was determined, values of 24,000 and 27,000 being obtained. A possible interaction of the... 

Fig. 4. Tantalum distillation columns and appropriate temperatures (K) for the preparation-purification of Am and Cm metals.

Online sample preparation Purification of gases by GC column Small sample size (micrograms)... 

When intermediate 274 reacted with ketones 133 (from D-glucose) or 270 (from D-fructose), mixed steroid-sugar compounds 278 and 279 were, respectively, obtained in low yields (10-16%). In addition, the use of protected estrone 280 (precursor of the epoxide 272) as electrophile allows the preparation of the dimeric steroid 281 in 26% yield. The low yields obtained for compounds 278, 279 and 281 are due to extensive decomposition during their column chromatographic purification. 

Hernandez, R., Nelson, S., Salm, J. R., Brown, D. T., and Alpert, A. J., Rapid preparative purification of West Nile and Sindbis virus PCR products utilizing a microbore anion-exchange column. Journal of Virological Methods 120(2), 141-149, 2004. 

The simplest and often the most cost effective way to combat friction is to reduce flow rate to a minimum. By no coincidence, this often leads to an increase in the efficiency of a separation since in many circumstances for preparative purifications, the less experienced have followed a linear scale-up from analytical column flow rates. In an ideal world each separation should, at some stage, involve a flow rate optimization. The fundamental principles behind this are discussed by JJ van Deemter[52 in what is probably the most cited paper in the history of chromatography. In summary, this suggests doing a graphical plot of separation efficiency versus flow rate and is particularly important for peptide purification where mass transport is comparatively slow. The van Deemter equation in simplified form can be represented as ... 

Fig. 5.10. Semi-preparative purification of 4-hydroxynonenal dinitrophenylhydrazone in microsomal fraction from 0.5 g rat liver. Reversed-phase column, Lichrosorb Si60 (51 m) solvent dichloromethane (50% in water flowrate 1.5 ml/min detection 340 nm injection 100-200 /il (a) Standard (b) Liver microsomal fraction. (Benedetti et...

NP-HPLC Normal-phase liquid chromatographic methods applying Diol-columns or common silica columns are well suited for the analysis of the total steryl ferulate content. They require very little sample preparation, as total lipid extracts can frequently be directly injected into the column without purification or fractionation. Run times for SFs are also relatively short, and a good separation from other lipid components can be obtained in less than 10 min in traditional HPLC systems. Depending on the column type and the sample, SFs elute as one or two peaks. Two peaks are obtained from the separation of SFs, which have ferulic acid both in cis- and trans- configuration (Nystrom et ah, 2008). The relative retention time (obtained with a silica column and hexane/ethyl acetate 97 3 as eluent) of the cis- form is about 0.5 smaller than that of steryl irans -ferulates (Akihisa et al., 2000). 

Fig. 13. Elution profile of the preparative purification of 1 g of L-Leu-Gly-Gly-Gly. Chromatographic conditions column, PrepPak-SOfl/Cu cartridge mobile phase, 95% water-5% methanol-0.05% trifluoroacetic acid, pH 2.3 flow rate, 100 ml/min. The arrows indicate where fractions were pooled fraction 5 was recycled once and the desired fraction 5a was collected. Reprinted with permission from Bishop ct al. (103). Copyright by Elsevier Scientific Publishing Co.. Amsterdam.

In 1972 Ogawa and Toyama (56) purified three components— A-I-a, A-I-b, and A-II-1—which were adsorbed on a gauze column during purification from Cellulase Onozuka P1500, a commercial preparation of T. viride cellulase. These three components had molecular weights of 32,000, 48,000, and 48,000 as determined by gel filtration and contained 7-16% carbohydrate. Each is reported to carry out the random hydrolysis of CM-cellulose and to degrade hydrocellulose (Avicel) and cellooligosaccharides except for cellobiose. The order of reactivity toward either cotton or Avicel was A-II-1 > A-I-b > A-I-a. The proteins adsorbed on cellulose comprised 38% of the total cellulase protein. 

The classical column procedures for the preparation of lysozyme are difficult to use with high viscosity solutions such as serum. A batch method was developed based on affinity chromatography using deami-nated chitin [82]. The method was found to give a one-step purification of nearly theoretical amounts in tissue homogenates tested. These included tissue homogenates from humans, primates, avian egg white and plants. The use of a chitin-coated cellulose affinity column for purification of lysozyme has also been described [83]. 

The purification of liprotein lipase (LPL) from rat adipose tissue [84] and hen adipose tissue [85], respectively, has been described using an affinity column prepared by insolubilization of heparin on agarose. In the case of hen adipose LPL elution was achieved with 1.16 M NaCl and 50% recovery achieved with 80-fold purification. 

As an example of the steps involved in affinity chromatography, we will consider the purification of an enzyme present in a mixture of proteins, using an immobilized competitive inhibitor as an affinity ligand. The first step after column preparation is the application of the sample during this step, the enzyme that binds to the inhibitor is retained, or adsorbed on the column. This is followed by a rinse step, whereby all nonbinding species are removed. The third step involves elution, and this step... 

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