Waters Associates column method

Figure 3. Separation of cellooligosaccharides using the Waters Associates column method (18). The solvent system employed was acetonitrile-water (72 28) at a flow rate of 4,0 mL/min,...

On-line size exclusion chromatographic (SEC) analyses were performed with a Waters Model 401 differential refractometer (DR), a Waters Model 480 ultraviolet (UV) variable wavelength spectrophotometer and a Foxboro Miran lA infrared (IR) photometer, equipped with a zinc selenide ultramicro flowcell of 1.5 mm nominal pathlength and 4.5 /xl volume, purchased from the same supplier. A set of ten Mycrostyra-gel (Waters Associates) columns, regenerated by Analytical Sciences Inc. (ASI) and of nominal porosities 100, 500 (two) 10 (two), 10 (three), 10 and lO X, in the order given and a mobile phase flow rate of 1 ml/min was used. The column set had a specific resolution of 19.7 in 1,4-dioxane, as determined by the method of Yau(2). 

Methylation analysis was run by the method of Hakomori ( ), followed by hydrolysis with trifluoracetic acid, sodium borohydride reduction, and acetylation. 6LC was performed on a Hewlett-Packard 5970, used as an inlet for a mass spectrometer. Molecular weight was determined on a Sephacryl S-500 column (2.6 x 70 cm), using deionized water as solvent, upward flow, 2.75 ml/min, and detection by refractive index monitor. Model R-401 (Waters Associates). 

In the initial phases of the analytical method development a Waters Associates C-jq y-Bondapak column was used. With certain dyes, such as C.I. Direct Black 38, aniline is used as a terminal group (Figure 1). This column caused the benzidine peak of the reduced dye to obscure a peak due to aniline. If these two compounds were not resolved, the method could not differentiate between benzidine-based and aniline-based dyes. This problem was removed by use of the Waters Radial Compression Module Model RCM 100 with a Radial Pak A cartridge. 

Column design and preparation incorporated previously described methods reported in the literature (39). Two different adsorbents were employed a 100/120 mesh crosslinked styrene/ divinylbenzene resin (Polypak P-Waters Associates) and a Woelm aniontropic activity grade alumina. These adsorbents were packed in 300 and 94 cm. stainless steel columns having a 1 mm. internal diameter. Pressure drop across the adsorbent bed was kept to a minimum (<0.02 atm.) by using a heated pressure reduction valve at the end of the column. Typical linear flow velocities through the columns were in the range of 0.27-2.17 cm/sec. 

HPLC Experiments. The liquid chromatograph model ALC 202 of Waters Associates fitted with a differential refractometer was used in this work. The method of column preparation and the general experimental technique used were the same as those reported earlier (2). All experiments were carried out at the laljoratory temperature (23-25°C). The solvent (water) flow rate... 

Sugars. The high performance liquid chromatography (HPLC) method described by Hunt et al. (8) was used to determine the sugars in the kiwifruit and nectars with some modifications (21). A Waters Association Chromatograph equipped with a Model 6000-A solvent delivery system, a Model R401 refractometer detector, a D6K universal injector column was a 30 cm x 4 mm i.d. stainless steel tube packed with u-bondapak-carbohydrate (Waters Associates). The precolumn was packed with CO-PELL PAC (Whatman). The eluent was acetonitril and distilled water (85/15, v/v). 

The packing material of the gas chromatography column was poropak Q (Waters Associates, Inc.). Details of the adsorption equilibrium measurement by the batch method can be found in Ma and Lin (12)... 

Haney et al [1,2], Waters Associates [3], and Knox et al. [4,5] - to name just a few - found that by adding lipophilic ions such as alkanesulfonic acid or quaternary ammoniiun compoimds to the mobile phase, solute ions of opposite chaise can be separated on a chemically bonded reversed-phase column. The term reversed-phase ion-pair chromatography (RPIPC) has generally been adopted for this technique. The term mobile-phase ion chromatography (MPIC) describes a method that combines the major elements of RPIPC with suppressed conductivity detection. Besides the above-mentioned chemically bonded reversed phases, neutral divinylbenzene resins featuring a high surface area and a weakly polar character are also used as stationary phases. 

Separations by HPLC were carried out using a Waters Associates Model 510 system with a Pharmacia Mono-Q anion-exchange column. Gradient elution (10 to 70% In 15 min, 1.5 ml/mln, 8 min before run) was performed with 20 mM piperazine buffer, pH 6.0, containing 20% Isopropanol (solvent A) and the same buffer containing 20% Isopropanol and 1.0 mM NaCl (solvent B). Subcellular localization studies were accomplished using the methods previously described (7). 

Elemental composition Cr 52.00%, 0 48.00%. The compound may he identified from its dark red color. Other color phases are noted above. Chromium may he measured in the aqueous phase hy AA, ICP or x-ray techniques, or in the solid phase hy x-ray methods. Hexavalent chromium (Cr6+) may he analyzed hy ion chromatography. For this, the aqueous sample is adjusted to pH 9 to 9.5 with a concentrated buffer (ammonium sulfate and ammonium hydroxide mixture) and mixed into the eluent stream of the buffer. Cr " is separated from Cr + on a column, and derivatized with an azide dye as a colored product measured at 530 nm, which is identified from its retention time. (APHA, AWWA, and WEF. 1999. Standard Methods for The Examination of Water and Wastewater, 20th ed., Washington, DC American Public Health Association.)... 

Elemental composition H 3.73%, C 44.44%, N 51.83%. HCN may be analyzed by GC or GC/MS. The aqueous solution may be directly injected onto the GC column and determined by an FID. For GC/MS determination, an alcoholic solution may be injected into the column. The characteristic mass ions are 27 and 26. The cyanide ion in aqueous solution also may be measured by cyanide ion-selective electrode, titrimetry, and by colorimetric methods (APHA, AWWA, WEF. 1999. Standard Methods for the Examination of Water and Wastewater, 20th ed. Washington, DC American Public Health Association). For colorimetric analysis, the aqueous solution may be treated with a dilute caustic soda solution, followed by treatment with chloramine-T, and then with pyridine-barbituric acid reagent. A red-blue color develops, the absorbance of which is measured by spectrophotometer at 578 nm. The concentration of CN is determined from a standard cahbration curve using KCN standards. 

Miwa and Yamamoto (31) described a simple and rapid method with high accuracy and reliability for the determination of C8 0-C22 6 fatty acids, which occur in esterified forms in dietary fats and oils and in living cells [the biological effects of routinely consumed fats and oils are of wide interest because of their impact on human health and nutrition (28,29), in particular, the ratio of cu-3 polyunsaturated fatty acid to w-6 polyunsaturated fatty acids (w-3/cu-6) seems to be associated with atherosclerosis and breast and colon cancers (30)]. They report improved separation of 29 saturated and mono- and polyunsaturated fatty acids (C8-C22), including cis-trans isomers and double-bond positional isomers, as hydrazides formed by direct derivatization with 2-nitrophenylhydrazine hydrochloride (2-NPH HC1) of saponified samples without extraction. The column consisted of a J sphere ODS-M 80 column (particle size 4 /xm, 250 X 4.6-mm ID), packed closely with spherical silica encapsulated to reach a carbon content of about 14% with end-capped octadecyl-bonded-spherical silica (ODS), maintained at 50°C. The solvent system was acetonitrile-water (86 14, v/v) maintained at pH 4-5 by adding 0.1 M hydrochloric acid with a flow rate of 2.0 ml/min. Separation was performed within only 22 min by a simple isocratic elution (Fig. 6). The resolution of double-bond positional isomers, such as y-linolenic ( >-6) and a-linolenic acid ( >-3) hydrazides and w-9, >-12, and >-15 eicosenoic acid hydrazides was achieved by use of this column. 

Concentration. Clarified filtrates, centrates, or column eluates are usually too dilute for use in their specific applications, thus, substantial amounts of water must be removed. This can be achieved by evaporation or by ultrafiltration. Concentration methods used in industrial settings, such as evaporation, which is done under vacuum, and solvent extraction, may or may not be suitable for dewatering proteins because of their potential for thermal or chemical denaturation, and due to high energy costs associated with evaporation. The benefit of evaporation is that nonvolatile compounds that may stabilize the proteins are retained. 

---