Sample Extraction and Clarification

Minimise sample handling Minimise use of additives Remove damaging contaminants early 

Definition Primary isolation of target protein from source material. 

Goal Preparation of a clarified sample for further purification. Removal of particulate matter or other contaminants which are not compatible with 

If sample extraction is required the chosen technique must be robust and suitable for all scales of purification likely to be used. It should be noted that a technique such as ammonium sulphate precipitation, commonly used in small scale, may be unsuitable for very large scale preparation. Choice of buffers and additives must be carefully considered if a purification is to be scaled up. In these cases inexpensive buffers, such as acetate or citrate, are preferable to the more complex compositions used in the laboratory. It should also be noted that dialysis and other common methods used for adjustment of sample conditions are unsuitable for very large or very small samples. 

For repeated purification, use an extraction and clarification technique that is robust and able to handle sample variability. This ensures a reproducible product for the next purification step despite variability in starting material. 

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The need for sample preparation prior to the first chromatographic step is dependent upon sample type. In some situations samples may be taken directly to the first capture step. For example cell culture supernatant can be applied directly to a suitable chromatographic matrix such as Sepharose Fast Flow and may require only a minor adjustment of the pH or ionic strength. However, it is most often essential to perform some form of sample extraction and clarification procedure. 

The final purification process should ideally consist of sample preparation, including extraction and clarification when required, followed by three major purification steps, as shown in Figure 2. The number of steps used will always depend upon the purity requirements and intended use for the protein. 

For extraction and clarification at laboratory scale Partially purifies sample, may also concentrate Use before the first chromatographic step... 

Unlike clenbuterol, salbutamol is a difficult compound to analyze due to its particular chemical attributes. It is a basic compound subjected to protein binding poor recoveries are obtained especially when protein precipitation techniques are used to prepare the extracts (145). In addition, salbutamol is charged at all pH values and does not readily lend itself to simple, specific back-extracting procedures. This severely restricts the options of sample cleanup. However, a Subtilisin protease digestion step followed by acid clarification and solid-phase extraction has been suggested (146) as an adequate extraction and cleanup procedure prior to the end-point determination of salbutamol by an enzyme immunoassay (139) based on the cross-reactivity of anticlenbuterol antibodies. 

Solvent Extraction Experiments. Solvent extraction studies were done on two feed samples representing dissolved hydroxide cake (SSA) and evaporator supernate (SSB). SSA was prepared by dissolution of hydroxide cake with slow addition of concentrated HN03, adjustment of the final acidity to 0.5 N by addition of water and/or HN03, and clarification by filtration. To prepare SSB, some supernate liquid from the evaporator was titrated, acidity adjusted to 0.5 N by NaOH and water addition, then clarified by filtration. No appreciable solids were observed on the clarification filters for either solution. Compositions of these feeds are listed in Table III. 

Abstract The clarification of hydrocarbon input into the Baltic sea via rivers is one of the priority issues of the 4th Pollution Load Compilation (PLC-4) within the framework of international Baltic Sea marine monitoring. An interlaboratory comparison was conducted to check the applicability of a new method for the determination of hydrocarbons by solvent extraction and gas chromatography. Surrogate oil solutions with known hydrocarbon content were distributed among the participants for preparation of water samples of different hydrocarbon con-... 

Over the years numerous changes to the extraction process have been proposed to improve the efficiency of nitrite extraction and eliminate interferences in the color-developing reaction of the various exogenous or endogenous compounds (ascorbic acid, fat, protein, etc.) present in biological samples and beverages. Most purification processes are based on deproteination and clarification of the extract, for which several types of agents have been used, mainly borax (sodium tetraborate anhydride), potassium aluminum sulfate, mercuric chloride, zinc sulfate, or potassium hexacyanoferrate. One of the... 

Figure 7. The effect of the mixing order in the BPA treatment protocol on the clarification parameters. Results are expressed as a percentage of those for the untreated sample. All samples contained 300 mM KCL BPA-1000 wasusedata concentration of 4000 ppm and BPA-1050 was used at a concentration of 6000 ppm. Legend = BPA-1000 treated extract, BPA added last El = BPA-1050 treated extract, BPA added last = BPA-1000 treated extract, extract added last Q = BPA-1050 treated extract, extract added last...

Clarification of extracts can be accomplished with Carrez reagents (44). Purification has been performed by solid phase or partition. Wu et al. (47) absorbed saccharin onto ODS-4 cartridges and then eluted with methanol phosphate buffer. Puttemans et al. (28,50) extracted saccharin from soft drinks and yogurt by ion-pair extraction with tri-n-octylamine and back-extraction to an aqueous phase with perchlorate. Tereda and Sakabe (48) used cetrimide and Sep-Pak Cl8 in the cleanup of saccharin in coffee drink. The column was preconditioned with methanol, water, and 5 mM cetrimide. The sample, diluted in phosphate buffer pH 3.0 containing cetrimide was poured into the cartridge, washed with water, and eluted with acetonitrile water (1 1, v/v). Moriyasu et al. (40) added n-propylammonium bromide to the extract, passed it through a Bond Elut Cl 8 column, and eluted the sweetener with a mixture of methanol-water (4 6, v/v). The eluate was passed through a Bond Elut SAX column and washed with 0.5% phosphoric acid and water, and the sweetener was eluted with 0.3 N hydrochloric acid. 

Clarification with Carrez solutions can be used to eliminate interfering compounds (14,44,45). Purification, isolation, or concentration of the extract can also be performed by solid-phase extraction with C8 (53) or Cl8 (46,47,75). Hayakawa et al. (75) used Sep-Pak Cl8 cartridges for the separation of aspartame from its degradation products. The sample was applied to the Sep-Pak, and degradation products and aspartame were eluted with 10% and 30% methanol in acetate buffer, respectively. 

The process of purification, also called downstream processing, depends on the product and the degree of purification required.74 Current strategies used for purification of therapeutic proteins generally involve these steps (1) sample preparation (clarification or extraction), (2) product capture (product concentration), (3) intermediate purification (removal of bulk impurities), and (4) polishing (removal of trace impurities) as shown in Figure 32.4. 

Soft drinks can be analyzed by direct injection, although sometimes, clarification or degasification is needed. For most complex samples, such as jams, sweets, or desserts, sample preparation comprises homogenization, extraction, cleanup, and concentration. 

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