Sample preparation ultrafiltration

Ultrafiltration processes (commonly UF or UF/DF) employ pressure driving forces of 0.2 to 1.0 MPa to drive liquid solvents (primarily water) and small solutes through membranes while retaining solutes of 10 to 1000 A diameter (roughly 300 to 1000 kDa). Commercial operation is almost exclusively run as TFF with water treatment applications run as NFF. Virus-retaining filters are on the most open end of UF and can be run as NFF or TFF. Small-scale sample preparation in dilute solutions can be run as NFF in centrifuge tubes. 

Until this point, the sample preparation techniques under discussion have relied upon differences in polarity to separate the analyte and the sample matrix in contrast, ultraflltration and on-line dialysis rely upon differences in molecular size between the analyte and matrix components to effect a separation. In ultrafiltration, a centrifugal force is applied across a membrane filter which has a molecular weight cut-off intended to isolate the analyte from larger matrix components. Furusawa incorporated an ultrafiltration step into his separation of sulfadimethoxine from chicken tissue extracts. Some cleanup of the sample extract may be necessary prior to ultrafiltration, or the ultrafiltration membranes can become clogged and ineffective. Also, one must ensure that the choice of membrane filter for ultrafiltration is appropriate in terms of both the molecular weight cut-off and compatibility with the extraction solvent used. 

Many process mixtures, notably fermentations, require sample preconcentration, microdialysis, microfiltration, or ultrafiltration prior to analysis. A capillary mixer has been used as a sample preparation and enrichment technique in microchromatography of polycyclic aromatic hydrocarbons in water.8 Microdialysis to remove protein has been coupled to reversed phase chromatography to follow the pharmacokinetics of the metabolism of acetaminophen into acetaminophen-4-O-sulfate and acetaminophen-4-O-glucu-ronide.9 On-line ultrafiltration was used in a process monitor for Aspergillus niger fermentation.10... 

A system based on microdialysis coupled with flow-injection chemiluminescence detection allows for direct sampling of unbound drug without extractive sample preparation [72], A similar approach based on continuous ultrafiltration has also been reported [73]. Modifications designed to overcome challenges of low solubility and high-non-specific binding in the ultrafiltration approach have also been described [74]. 

Both column chromatography and HPLC are used routinely for sample preparation, particularly for protein samples after particulate contamination has been removed by filtration or centrifugation. In addition, the use of ultrafiltration or solid-phase extraction techniques prior to chromatography often will result in a simplified, more concentrated sample. 

After the introduction of pronase E, other more or less nonsubstrate-specific proteolytic enzymes have been applied to assist Se speciation. Most of them were derived from DNA/RNA clean-up protocols. The new enzymes (subtilisin from Bacillus licheniformis, also named protease VIII, EC 3.4.21.14 proteinase K from Tritirachium album, EC 3.4.21.64 the crude Novo Nordisk product of Flavourzyme from Aspergillus oryzae) proved to be capable of extracting Se with varying yields and chromatographic recovery of Se species. It is important to highlight that the latter parameter also depends on the instrumentation available. In this regard, different recovery values for the same samples reported by independent research groups do not necessarily indicate successful or unsuccessful sample preparation. Similarly, extraction efficiency (defined as the ratio of extracted Se to total Se in the sample) cannot be used as such for comparison purposes because sample preparation may include some extra steps, for example, TCA precipitation or ultrafiltration, which may reduce this value even by 10-20 percent. 

Thormann et al. (1993) have published an overview of the strategies for using MEKC to monitor drugs in body fluids (serum, urine, saliva) they discuss buffer selection and sample preparation (direct injection, ultrafiltration, solid phase extraction. 

The technique of ultrafiltration for serum sample preparation has been studied by several investigators (H7, HIO, K9, S19, V4, V5). In this technique the serum sample is passed through an exclusion membrane which exhibits a 95% retention for compounds with MW > 25,000. Thus, low-molecular-weight constituents pass through the membrane, whereas proteins or protein-bound constituents are retained. Hartwick ct al. (HIO) reported excellent recoveries for xanthosine, inosine, guanosine, theobromine, theophylline, and caffeine. Tryptophan showed poor quantitative recovery in accordance with the findings of previous investigators (K33, 01). 

Atomic force microscopy. Scanning electron microscopes do not provide the necessary resolution for analyzing detail morphology of certain membrane layers of very fine pore sizes such as those suitable for ultrafiltration and gas separation. While transmission electron microscopes are capable of examining very small scale structures, the technique is limited to only very thin specimens. This makes sample preparation for a multilayered membrane very difficult and tedious. 

Two additional techniques that are used in sample preparation protocols are ultrafiltration and microdialysis. In ultrafiltration, pressure is applied to a membrane and those molecules smaller than the mo-... 

An extensive examination of the different conditions affecting CZE separation (sample preparation, type of loading buffer and pH of CE buffer) of wine proteins reveals as optimum conditions the sample preparation by either ultrafiltration or dialysis and dissolution and separation... 

To analyze free amino acids in plasma or tissue homogenates, it is necessary to remove proteins and peptides present in solution. The most widely used deproteinization method is precipitation with 5-sulfosalicylic acid followed by centrifugation for separating the precipitate. In comparison to other precipitation agents such as trichloroacetic acid, perchloric acid, picrinic acid, or acetonitrile, the best results with respect to completeness of precipitation are obtained with 5-sulfosalicylic acid [39]. Other deproteinization methods comprise ultrafiltration and ultracentrifugation [40], which have only recently been considered as sample preparation methods for amino acid analysis. 

Certain techniques are very good at particular tasks (such as protein precipitation for removal of proteins and cellular components), but perhaps not as good in general. With the emphasis on greater efficiency, many of these approaches have become automated or semiautomated. Thus, there has been greater emphasis on the 96-well formats an approach that can lend itself more readily to automated workstations. This format has been used successfully for protein precipitation, liquid—liquid extraction, and solid-phase extraction [8-10]. Ultrafiltration (UF) in a 96-well format is also being evaluated and shows some potential, but products and applications are not yet fully developed. Automated techniques for sample preparation and each of the sample preparation techniques listed in Table 1 are described below. 

In principle, ultrafiltration can be an easy way to quantify free drug fraction present in plasma, serum, or other biological fluids. The approach is not without pitfalls, however, in that ion suppression, clogged membranes, and poor sensitivity due to extensively bound drugs can derail this type of assay. Still, ultrafiltration has a lot of untapped potential and could become pervasive as membranes are made more robust and adapted to high-throughput formats such as 96-well plates. A related sample preparation technique, microdialysis, is discussed in Chapter 12. 

T vo approaches can be adopted for dealing with the overlap problem. Firstly, simple sample preparation such as centrifugal ultrafiltration can be used to remove the macromolecules. This results in a spectrum of all the nonprotein-bound metabolites contributing to the spectrum. Alternatively, to avoid s unple manipulation, a spectral editing technique can be applied. It has... 

Sample preparation Filter 1 mL broncho-alveolar lavage fluid (Tosoh Ultracent-30 with a molecular mass cut-off at 30000) while centrifuging at 1500 g at 5° for 30 min, inject a 100 (xL aliquot of the ultrafiltrate. 

Sample preparation 500 xL Milk + 500 p,L MeCN MeOH water 40 20 40, vortex for 10-15 s, filter (Centricon-10, molecular mass cut-off filter 10000 daltons) with centrifuging at 2677 g for 30 min, iiyect a 10-100 p,L aliquot of the ultrafiltrate. 

Sample preparation Serum. Filter using Molcut II (Millipore), inject a 50 xL aliquot of the ultrafiltrate. Urine. Dilute ten-fold with water, filter ((Telman acrylate copolymer 0.45 pm), inject a 20 pL aliquot of the filtrate. 

Sample preparation Plasma. Filter (Amicon MPS-1 with a YMT membrane) while centrifuging at 3000 g at 4° for 15 min, inject an aliquot of the ultrafiltrate. Urine. Inject tm aliquot directly. 

Sample preparation Separate buffer containing drug from human serum albtunin by centrifuging at 37° at 700 g for 3 min using a Micropartition System MPS-1 (Amicon) unit, inject a 10-20 pL aliquot of the ultrafiltrate. 

Sample preparation 500 p,L Serum + 500 p,L MeCN EtOH water (40 40 20), vortex for 10-15 s, centrifuge through a Centricon-10 fflter unit with a 10000 dalton cut-off (Amicon) at 4000 g for 30 min, inject a 10-60 p,L aliquot of the colorless ultrafiltrate. 

Sample preparation Blood. Hemolyze 25 p,L whole blood with 50 pL water. 25 p,L Plasma or hemolyzed blood + 100 pL 100 pg/mL ranitidine + 100 xL 5 M NaOH + 5 mL dich-loromethane, mix, shake for 10 min, centrifuge at 1650 g for 10 min. Remove 4 mL of the organic layer emd evaporate it to dryness, reconstitute the residue in 100 p-L mobile phase, inject a 25 pL aliquot. (To measime unbound cimetidine in plasma inject 25 pL ultrafiltrate (Amicon MPS-3 centrifree).) Tissue. Brain tissue + 100 pL 50 pg/mL ranitidine + 1 mL saline, homogenize in an ice bath for 1 min, add 100 pL 1 M NaOH, add 5 mL dichloromethane, extract. Remove 3 mL of the organic layer and evaporate it to dr3mess, reconstitute the residue in 100 pL mobile phase, centrifuge at 10000 g, iiyect a 25 pL aliquot. CSF. Inject an aliquot directly. 

Sample preparation Urine. Add 10 pL urine diluted 10 times with 10 mM pH 7.5 Na2HP04 buffer to 1-5 pg nizatidine, make up volume to 300 pL with 10 mM pH 7.5 Na2HP04 buffer. Place solution on YM-10 ultrafiltration membrane with a cut-off of 10000, centrifuge at 4000 g for 20 min. Mix 180 pL filtrate with 20 pL MeOH, inject 50 pL. Perfusate. Add 10-100 pL perfusate to 1-5 pg nizatidine, make up volume to 300 pL with 10 mM pH 7.5 Na2HP04 buffer. Place solution on YM-10 ultrafiltration membrane with a cut-off of 10 000, centrifuge at 4000 g for 20 min. Mix 180 pL filtrate with 20 pL MeOH, inject 50 pL. 

Sample preparation Serum. Wash Amicon YMB filter membrane by stirring gently in 200 mL 100 mM pH 7.0 sodium phosphate buffer for 30 min, blot dry with filter paper. Dilute serum with an equal volume of 100 mM pH 7.0 sodium phosphate buffer, filter (Amicon YMB) while centrifuging at 5° at 1000 g for 15 min. Add 1 part reagent to 4 parts ultrafiltrate, let stand for 10 min, iiyect a 25-50 pL aliquot. Urine. Dilute 10-fold with 100 mM pH 7.0 sodium phosphate buffer. Add 1 part reagent to 4 parts diluted urine, let stand for 10 min, inject a 25-50 p,L aliquot. (Reagent was 8.25 g imidazole, 24 mL water, and 2 mL 5 M HCl made up to 40 mL with water.)... 

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