Columns gradient

Figure 4. Separation of ribonucleoside monophosphofic acids. Conditions 250-cm anion exchange column gradient, 0.01M KHgPO containing HsPOi, (pH 2.6) to 0.15M KHiFO in 30 min column tempera-ture, 70 C detector, UV at 254 nm. 1, cyti-dine-S -monophosphoric acid 2, uridine-5 -monophosphoric acid 3, adenosine-5 -mon-ophosphofic acid 4, inosine-5 -monophosphoric acid 5, 3, 5 -cyclic adenosine mono-phosphoric add 6, guanosine-5 -monophosphoric acid (36).

The MRM experiments do not require chromatographic separation of the metabolites. Therefore, other LC conditions, columns, gradient, and injection volumes may be used provided that there is adequate sensitivity and specificity, and the chromatographic quality is not compromised. 

Murren, C., Stelling, D., and Felstead, G., An improved buffer system for use in single-column gradient-elution ion-exchange chromatography of amino acids, /. Chromatogr., 115, 236, 1975. 

By replacing conventional 3.5 or 5 jtm columns with sub-2-micron columns, gradient time can be reduced dramatically. The flow rate must be increased for optimal conditions as well but solvent consumption will be less than the amount used by the original method. To use the full power of these columns, an LC instrument must be thoroughly optimized toward lowest extra-column dispersion. The smaller the column (small ID and short length), the more sensitive the performance is to dispersion. With smaller internal diameter columns, the injection volumes and internal diameters of the capillaries should be reduced. 

The submitters followed the course of the reaction by HPLC, monitoring for the disappearance of 4-chlorophenyl trifluoromethanesulfonate. HPLC conditions were as follows 0.46 x 7.5 cm Eclipse-XDB column, gradient elution (10 80 85 15 acetonitrile/10 mM pH 6.3 phosphate buffer over 8.5 min, flow rate = 1,5 mLVmin, detection = 210 nm). Rj (4-chlorophenyl trifluoromethanesulfonate) = 6.25 min, Rj (n-butyl 4-chlorophenyl sulfide) = 7.10 min. 

Thesenon-Emeric, G. and F.E. Regnier "Process Monitoring by Parallel Column Gradient Elution Chromatography. . -Statistical (in-on Sit".. 11 ]4 (June I. 1991). Wahl. J.H.. C.G. Eskc. and V.L. McGufTin "Solvent Modulation in Liquid t hru maiography." Analytical Chemistry. 1117 (June I. 1991). 

Fig. 5. Gradient elution of standard proteins on a Zorbax PSM C 18 column. Gradient 0.01 M phosphate buffer pH 2/acetonitrile samples 1 — ribonuclease A, 2 — insulin, 3 — cytochrome C, 4 — lysozyme, 5 — transferrin, 6 — serum albumin, 7 — ovalbumin. (From Ref. 151 with permission)...

Fig. 9 Chromatogram of the separation of the dabsyl derivatives of all 20 naturally occurring amino acids on a Cik column. Gradient elution employed acetonitrile, methanol, and acetate buffer (pH 6.5). Note the presence of a large peak due to the hydrolysis product of excess derivatizing reagent. Peaks marked with asterisks have not been identified. (From Ref. 154. Copyright 1991 Elsevier Science.)...

To determine a small amount of His in sparkling wine, Tarrach proposed a method based on SPE with an Amberlite GC-50 column, derivatization of the eluate with PA, and HPLC analysis on a Supershere 100 RP-18 column (gradient elution with ACN-acetate buffer). Fluorimetric detection at 450 nm allowed DLs of 0.05 -2.00 mg/L (128). 

Aqueous samples may be analyzed by HPLC on an underivatized polysty-rene-divinylbenzene column such as PolyRPCO (Alltech 1995) or a C-18 reverse phase column gradient acetonitrile and 0.01 M K3P04 at pH 7 (55 45) and the analyte detected by UV at 254 nm. 

Fig. 1.22. Direct injection of aluminium processing solution. Conditions Supelcosil LC-18-DB column gradient programme at a flow-rate of 1.0 ml/min from 1.05 M HIBA to 0.4 M HIBA over 10 min. and held at 0.4 M for 5 min. modifier, 1-octanesulphonate at 0.01 M (A), eluents at pH 4.5 (B), eluents at pH 3.8 detection at 658 nm after post-column reaction with Arsenazo III sample injected, 50 pi sample dilution, (A) 10 ml to 100 ml,...

Fig. 1.24. Effect of pH on the retention time of the rare earths. Conditions Supelco LC-18 DB column gradient program at a flow rate of 1.0 ml/min from 0.05 mol/1 HIBA to 0.4 mol/1 HIBA over 10 min and held at 0.4 mol/l for 5 min modifier, concentration of 1-octanesulfonate at 1 x 10 -.

After optimization of the method, the following analytical conditions were used silica capillary with stationary phase HP-5MS (30 m x 0.25 mm internal diameter, 0.25 pm film thickness) column gradient temperature started at 80 °C and increased at a rate of 10 °C/min to 280 °C injection temperature was fixed at 250 °C and helium was used as a carrier gas with constant flow rate of 1 mL/min. A mass spectrometry detector with single quadrupole was used as analyzer, operated at 150 °C with an acquisition scan rate of 50-800/n/z. 

Figure 4-13. Assembly of an ion exchange column, gradient mixer, and magnetic stirring motor. Arrows indicate points where the column is closed with pinch clamps.

Fig. 4 5 The analytical RP-HPLC chromatogram and the CE chromatogram of the purified AFP-Type III protein are shown. HPLC conditions VYDAC RP-18 column gradient 20/20/60% (H20/ACN/MeOH)+0.1%TFA into 30 min. to 0/10/90% into 35 min. 0/100/0%. The CE conditions are as discribed into the material section.

The second type is HPPLC (Table II). In HPPLC, the initial mobile phase is a nonsolvent for the sample copolymers, so that the sample copolymers injected into a column precipitate on the top of the column. Gradient elution is performed by adding a good solvent to the sample... 

Examples using HPLC include Spherisorb S5W silica column (4.9 mm i.d. x 125 mm), eluent - methanolic ammonium perchlorate (10 mM) plus 1 ml/L methanolic NaOH (0.1M), UV detection (254 nm), no electrochemical response, relative Rt reported 3 pBondapak C18 column, eluent -methanokphosphate buffer (2 3), ratio of UV absorbance (254 and 280 nm) measured, relative Rt reported 4 pBondapak RP-18 column (20 x 0.6 cm i.d.), eluent - methanokwater (7 3), UV detection (267 nm), Rt 5.3 min21 Zorbax C8 column, gradient elution using 0.1% H3PO4 and 0.1% H3P04./10% water in... 

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