Sodium phosphate buffer

All chemicals were used as received. PDADMAC and PAMPS were obtained from Aldrich Chemical Co. (Milwaukee, WI). Diclofenac sodium, sodium sulfathiazole, labetalol HCl, propranolol HCl, verapamil HCl, and diltiazem HCl were purchased from Sigma Chemical (St. Louis, MO). Dextrose USP was obtained from Amend Co. (Irvinton, NJ). Water was distilled and deionized using a Nanopure purihcation system (Fischer Scientihc, Fair Lawn, NJ). Simulated intestinal fluid was prepared using a O.OIM phosphate buffer (sodium phosphate monobasic and potassium phosphate dibasic) at pH 7 and 5.5 with different amounts of NaCl to vary the ionic strength. Simulated gastric fluid (pH 1.5) was prepared with concentrated HCl with different amounts of NaCl to vary the ionic strength. 

Figure 3.9 Measurement of the enantiomeric purity of the pharmaceutical intermediate SB-240093 using CD-modified CE. The electropherogram shows the analysis of the chiral system suitability standard containing 0.5% w/w of the R-enantiomer. (Conditions PVA-coated fused silica capillary, 50 cm effective length, 57 cm total length, 50 pm i.d. buffer sodium phosphate [pH 7.0, 100 mM] containing 1.75 mM dimethyl-/l-CD voltage —30 kV [reversed polarity] temperature 20°C detection UV at 200 nm sample preparation 0.5 mg/ml in water DMSO (95 5, v/v) sample introduction 6 s at 35 mbar, capillary inlet at cathode.)...

Buffer sodium phosphate, 50 mM, pH 8.0 I-labeled Bolton-Hunter reagent, 0.2 mCi in benzene-0.2% di-methylformamide (Amersham/Searle, > 1400 Ci/mmol)... 

Figure 2.11 Time course curves of the repeated UDP-Glc production using three enzymes (MalPase, GlPTTase, and PPase) immohilized on amino-functionalized magnetic nanoparticles. Reaction conditions MalPase, 18 U L GlPTTase, 100 U L PPase, 25 U L" maltodextrin, 5% (m/v) UTP, 2 mM MgCh, 10 mM buffer, sodium phosphate, pH 7.5, 100 mM 30 °C total volume, 200 mL.

FIGURE 2 Free solution separation of model peptides and tryptic digest of cytochrome c. Capillary 72 cm (50 cm to detector), 50- m id injection 1-sec vacuum analysis 200 nm, 30°C sample 100 g/mL each peptide in water, 100 g/mL tryptic digest buffer sodium phosphate (pH 2.5, 7.0), sodium acetate (pH 4.0), sodium tetraborate (pH 9.0), 20 mM in all cases. 

Proteins are precipitated by addition of ammonium sulfate to 73 % of saturation and storage during 3 hours. The proteins are collected by centrifugation 30 minutes at 9000xg and are resuspended with a minimum of chromatographic buffer (Sodium phosphate 10 mM, pH 7.3, 2-mercaptoethanol 8 mM, sodium azide 3 mM, glycerol 10 %). 

The ionic species of the mobile phase will also affect the separation. This is shown in Table 4.3 by the difference in resolution values for magnesium chloride buffer compared to sodium sulfate buffer. In addition, calibration curves for proteins in potassium phosphate buffers are shallower than those generated in sodium phosphate buffers. The slope of the curve in Sorenson buffer (containing both Na and ) is midway between the slopes generated with either cation alone (1). Table 4.4 illustrates the impact of different buffer conditions on mass recovery for six sample proteins. In this case, the mass recovery of proteins (1,4) is higher with sodium or potassium phosphate buffers (pH 6.9) than with Tris-HCl buffers (pH 7.8). 

Three different types of columns packed with gels of different pore sizes are available. Columns should be selected that are suitable for the molecular weight range of specific samples, as each type has a different exclusion limit (Fig. 6.41, page 215). Bovine serum albumin (BSA), myoglobin, and lysozyme show good peak shapes using only 100 mM of sodium phosphate buffer as an eluent. There is no need to add any salt to the eluent to reduce the ionic interaction between protein and gel. 

FIGURE 6.40 Calibration curves of Shodex PROTEiN KW-800 series. Column Shodex PROTEiN KW-802.S, KW-803, KW-804, 8.0 mm i.d. X 300 mm. Eiuent Pullulan, PEG PEO Purified water, Protein SO mM Sodium phosphate buffer + 0.3 M NaCI (pH 7.0). Flow rate 1.0 mL/min. Detector Pullulan. PEG PEO Shodex Rl Protein Shodex UV (220 nm). Column temp. Ambient. 

Equilibrate the column with 3 bed volumes of 20 mM sodium phosphate buffer, 300 mM sodium chloride, pH 7.2. 

FIGURE 7.10 Dependence of the resolution on the sample volume. A preparative Superformance column 1000-200 (bed volume 20 liters) packed with Fractogel END BioSEC (S) (bed height 63 cm) was loaded with 60 ml (top) and 300 ml of a mixture of bovine serum albumin (5 mg/ml), ovalbumin (5 mg/ml), and cytochrome c (3 mg/ml) (bottom) (20 m/VI sodium phosphate buffer, 0.3 M NaCI, pH 7.2 flow rate 100 ml/min corresponding to 19 cm/hr). When the sample volume is 300 ml the separation efficiency for BSA and ovalbumin is similar. Thus the column can be loaded with larger sample volumes, resulting in reasonable separations. 

FIGURE 7.13 Preparative separation of various proteins on Fractogel EMD BioSEC (S). The length of the column was 1000 mm and the inner diameter 100 mm. The flow rate was 6.2 ml/min with 20 sodium phosphate buffer (pH 7.2) containing 0.3 M NaCI as the eluent. The injected standard proteins can be used to create a calibration curve. 

FIGURE 7.17 Separation of a complex mixture on Fractogel EMD BioSEC (S) with a column dimension of 1000 X 50 mm (Superformance glass column). The sample contained ferritin (I), immunoglobulin G (2), transferrin (3), ovalbumin (4), myoglobin (5), aprotinin (6), and vitamin B, (7). Five milliliters of the mixture was injected onto the column at a flow rate of 3 ml/min (eluent 20 mAI sodium phosphate buffer, 0.1 M NaCI, pH 7.2). 

For many proteins, a simple buffer such as 0.1M phosphate, pH 7, produces excellent separations on SynChropak GPC columns. Generally, minimal interaction is achieved when the ionic strength is 0.05-0.2 M. To prevent denatur-ation or deactivation of proteins, the pH is generally kept near neutrality. For denatured proteins, 0.1% sodium dodecyl sulfate (SDS) in 0.1 M sodium phosphate, pH 7, is recommended. 

Solvent 50 mM sodium phosphate buffer, pH 7 Polymer concentration 0.1 to 0.5%... 

This is a crystalline product of insulin and an alkaline protein where the protein/insulin ratio is called the isophane ratio. This product gives a delayed and uniform insulin action with a reduction in the number of insulin doses necessary per day. Such a preparation may be made as follows 1.6 g of zinc-insulin crystals containing 0.4% of zinc are dissolved in 400 ml of water, with the aid of 25 ml of 0.1 N hydrochloric acid. To this are added aqueous solutions of 3 ml of tricresol, 7.6 g of sodium chloride, and sufficient sodium phosphate buffer that the final concentration is As molar and the pH is 6.9. 

Fig. 3-3. Comparison of the values of enantiomeric resolution of different DNP-D,L-amino acids at different deconvolution stages of a cyclic hexapeptide sublibrary. Resolution values in a cyclo(Arg-Lys-X-X-X-P-Ala) sublibrary, in the first line, are compared to those obtained in sublibraries with a progressively increasing number of defined positions. All the sublibraries were 30 mM in the running buffer while the completely defined cyclo(Arg-Lys-Tyr-P-Tyr-P-Ala) peptide is used at 10 mM concentration. Conditions cyclopeptide sublibrary in 20 mM sodium phosphate buffer, pH 7.0 capillary, 50 pm i.d., 65 cm total length, 57 cm to the window V = -20 kV, I = 40 electrokinetic injection, -10 kV, 3 s detection at 340 nm. (Reprinted with permission from ref. [75]. Copyright 1998, American Chemical Society.)...

Figure 2 Stability of /3-poly(L-malate) measured by its activity to inhibit purified DNA polymerase a of P. polyceph-alum. The relative degree of inhibition is shown (100 rel. units refer to complete inhibition). The DNA polymerase assay was carried out in the presence of 5 /tg/ml /S-poly(L-malate) as described [4]. The polymer was preincubated for 7 days at 4°C in the following buffer solutions (50 mM) KCl/HCl (—A—). Citrate (—V—). 2-(A/-Morpholino)-ethanesulfonic acid, sodium salt (—O—). Sodium phosphate (— —). N-(2-Hydroxyethyl)piperazine-N -(2-ethanesul-fonic acid), sodium salt (— — ). N,N-b s (2-Hydroxyethyl)-glycine, sodium salt (—T—). Tris/HCl (— —). 3-(Cyclo-hexylamino)-l-propanesulfonic acid, sodium salt (— —).

Reactions between aziridine-2-carboxylic acids and thiols in aqueous solution have been explored by Hata and Watanabe [112]. The reactions occurred predominantly at C-2 instead of C-3 to afford 3-amino acids, with the reaction between 148 (Scheme 3.53) and thiophenol in 0.2 m sodium phosphate buffer at room tem-... 

EDTA concentration and the degree of inhibition (Shimomura et al., 1961). In a 30 mM sodium phosphate buffer containing 60 mM NaCl, pH 7.0, inhibition by EDTA is very strong (84%) even at an EDTA concentration as low as 25 pM, but the inhibition does not increase beyond 90% even at an EDTA concentration of 5 mM. The inhibition is completely reversed by the addition of a sufficient amount of Ca2+ to bind EDTA. 

Fig. 3.1.4 Bioluminescence spectrum of Cypridina luciferin catalyzed by Cypridina luciferase (A), the fluorescence excitation spectrum of oxyluciferin in the presence of luciferase (B), the fluorescence emission spectrum of the same solution as B (C), and the absorption spectrum of oxyluciferin (D). The fluorescence of oxyluciferin alone and luciferase alone are negligibly weak. Measurement conditions A, luciferin (lpg/ml) plus a trace amount of luciferase in 20 mM sodium phosphate buffer, pH 7.2, containing 0.2 M NaCl B and C, oxyluciferin (20 pM) plus luciferase (0.2mg/ml) in 20 mM sodium phosphate buffer, pH 7.2, containing 0.2 M NaCl D, oxyluciferin (41 pM) in 20 mM Tris-HCl buffer, pH 7.6, containing 0.2 M NaCl. All are at 20°C.

Fig. 3.1.5 Effects of salt concentration on the activity of Cypridina luciferase (solid lines) and quantum yield (dotted lines). In the activity measurement, Cypridina luciferin (1 pg/ml) was luminesced with a trace amount of luciferase in 2.5 mM HEPES buffer, pH 7.5, containing a salt to be tested, at 20°C. In the measurement of quantum yield, luciferin (1 pg/ml) was luminesced with luciferase (20 pg/ml) in 20 mM sodium phosphate buffer (for the NaCl data) or MES buffer (for the CaCl2 data), pH 6.7.

Fig. 3.1.7 Effects of temperature on the activity of Cypridina luciferase (solid line) and the quantum yield of Cypridina luciferin (dashed line). Luciferin (1 pg/ml) was luminesced in the presence of luciferase (a trace amount for the activity measurement 20 pg/ml for the quantum yield) in 50 mM sodium phosphate buffer, pH 6.8, containing 0.1 M NaCl.

Johnson et al. (1962) measured the quantum yield of Cypridina luciferin in the luciferase-catalyzed reaction for the first time, using a photomultiplier calibrated with two kinds of standard lamps. The measurement gave a value of 0.28 0.04 at 4°C in 50 mM sodium phosphate buffer, pH 6.5, containing 0.3 M NaCl. The quantum yield... 

Fig. 3.2.2 Influence of pH on the initial light intensity of euphausiid luminescence when the fluorescent compound F and protein P are mixed in 25 mM sodium phosphate buffers of various pH values, each containing 1M NaCl, at near 0°C. Both F and P were obtained from Meganyctiphanes norvegica. From Shimomura and Johnson, 1967, with permission from the American Chemical Society.

Fig. 3.3.2 Influence of pH on the activity of luciferase ( ) and the quantum yield of coelenterazine (o) in the bioluminescence of Oplophorus. The measurements were made with coelenterazine (4.5 pg) and luciferase (0.02 pg) for the former, and coelenterazine (0.1 pg) and luciferase (100 pg) for the latter, in 5 ml of 10 mM buffer solutions at 24° C. The buffer solutions used sodium acetate (pH 5.0), sodium phosphate (pH 6.0-7.5), Tris-HCl (pH 7.5-9.1), and sodium carbonate (pH 9.5-10.5), all containing 50 mM NaCl. Replotted from Shimomura et al., 1978, with permission from the American Chemical Society.

Purification of Latia luciferase and the purple protein. According to Shimomura and Johnson (1968c), frozen specimens of Latia (10 g) are vigorously shaken in 200 ml of cold 5mM sodium phosphate buffer (pH 6.8) for 15 minutes. Latia luciferase is extracted into the buffer and the solution becomes turbid. Four batches of such turbid solutions are combined and centrifuged, and the clear supernatant is... 

Fig. 6.1.6 Effect of the purple protein on the luminescence of Latia luciferin (0.16 jxg) plus Latia luciferase (A280,icm 1.2, 10 pi) in 5 ml of 5mM sodium phosphate buffer, pH 6.8. The amounts of the purple protein solution ( 280,1 cm 0.6) used 20 pi (curve 1), 5 pi (curve 2), 1 pi (curve 3), 0.5 pi (curve 4), 0.2 pi (curve 5), and none (curve 6). From Shimomura and Johnson, 1968c, with permission from the American Chemical Society.

Fig. 6.1.7 Effect of pH on the initial light intensity and total light of Latia bioluminescence reaction in the presence of the purple protein, in 50 mM sodium phosphate buffer solutions having various pH values at 25°C (Shimomura et al., 1966b).

Purification of photoprotein. The dialyzed photoprotein solution was centrifuged to remove precipitates, and then subjected to fractional precipitation by ammonium sulfate, taking a fraction precipitated between 30% and 50% saturation. The protein precipitate was dissolved in 50 ml of 10 mM sodium phosphate, pH 6.0, containing 0.1 mM oxine ( pH 6.0 buffer ), dialyzed against the same buffer, and the dialyzed solution was adsorbed on a column of DEAE-cellulose (2.5 x 13 cm) prepared with the pH 6.0 buffer. The elution was done by a stepwise increase of NaCl concentration. The photoprotein was eluted at 0.2-0.25 M NaCl and a cloudy substance (cofactor 1) was eluted at about 0.5 M NaCl. The photoprotein fraction was further purified on a column of Sephadex G-200 or Ultrogel AcA 34 (1.6 x 80 cm) using the pH 6.0 buffer that contained 0.5 M NaCl. 

Step 2. The pellets are extracted with 10 mM sodium phosphate buffer, pH 6.7, containing 2mM EDTA and 0.2 M NaCl, and chromatographed on a gel-filtration column (Ultragel AcA 54 LKB) using the same pH 6.7 buffer. The photoprotein is eluted slightly before a brownish substance. 

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