Eluents trifluoroacetic acid

Figure 3.15 Chromatogram of fibre-type proteins on polystyrene gels having different pore sizes. Column A, PLRP-S 300 A, 15 cm x 4.6 mm i.d. B, PLRP-S 1000 A (polystyrene gel), 15 cm x 4.6 mm i.d. eluent, 15 min linear gradient from 20% of 0.25% trifluoroacetic acid to 60% of 0.25% trifluoro-acetic acid in 95% aqueous acetonitrile flow rate, 1.0 ml min-1 detection, UV220 nm. Peaks 1, collagen (Mr 120 000) and 2, fibrinogen (Mr 340 000). (Reproduced by permission from Polymer Laboratories data)...

Figure 3.23 Selectivity of phenyl and alkyl bonded stationary phase materials for protein separation. Column A, TSK gel phenyl-5PW RP, 75 mm x 4.6 mm i.d. B, TSK gel TMS 250, 75 mm x 4.6 mm i.d. eluent, 60 min linear gradient elution from 5% of 0.05% trifluoroacetic acid in 5%> aqueous acetonitrile to 80% of 0.05% trifluoroacetic acid in 80% aqueous acetonitrile flow rate, lml min-1 detection, UV 220 nm. Peaks 1, ribonuclease 2, insulin-, 3, cytochrome c 4, lysozyme-, 5, transferrin-, 6, bovine serum albumin-, 1, myoglobin-, and 8, ovalbumin.

Figure 25-16 Effect of sample solvent on retention time and peak shape of n-butylaniline. Eluent (1 mL/min) is 90 10 (vol/voh H20/acetonitrile with 0.1 wt% trifluoroacetic acid. Lower sample was dissolved in eluent. Upper sample was dissolved in methanol, which is a much stronger solvent than eluent. Column 15 cm x 4.6 mm, 5-ia.m-diameter Cle-silica, 30°C. Injection 10 iL containing 0.5 g analyte. Ultraviolet detection at 254 nm. [Courtesy Supelco, Belletonte, PA.]...

If you use a buffer in the eluent, it must be volatile or else it will evaporate down to solid particles that scatter light and obscure the analyte. Low-concentration buffers made from acetic, formic, or trifluoroacetic acid, ammonium acetate, diammonium phosphate, ammonia, or triethylamine are suitable. Buffers for evaporative light scattering are the same as for mass spectrometric detection. 

Denaturation in solution is known to be faster with 1-propanol than with 2-propanol 60), but in contact with the C 4 RP column a higher rate was observed with 2-propanol. Acetonitrile produced the highest acceleration. Replacement of H3P04 by 0.01 M trifluoroacetic acid did not alter the 2.2 pH, but enhanced by a factor of three the rate of denaturation at 22 °C. Thus, eluent systems containing TFA and... 

Mobile phase Eluent (A) acetonitrile eluent (B) water-trifluoroacetic acid, pH 2.5 Use 20-min gradient of 15-35% acetonitrile in water adjusted to pH 2.5 with TFA. 

Fig. 8.24. Separation of a tryptic cytochrome C digest by pressure assisted gradient CEC. Column, 60 x 0.18 mm i.d. packed with 3 pm Vydac Cl8 eluents, (A) 0.07% trifluoroacetic acid in water, (B) 0.07% trifluoroacetic acid in acetonitrile gradient elution with 0-50% B in 20 min applied pressure, 9 MPa in (a), 5 MPa in (b) 7 MPa in (c) applied voltage, 0 kV in (a), 1 kV in (b), 0.6 kV in (c) Detection, nanospray ESI-MS, m/z 200-1500, 10 spectra/s sample, tryptic digest of 8 pmol bovine cytochrome C. (Reprinted with permission from ref. [45], Copyright [1997] American Chemical Society).

The eluents most commonly used in peptide purification by reversed phase HPLC are water and acetonitrile. These are often buffered with trifluoroacetic acid (0.1% v/v, TFA), ammonium acetate (0.05-0.1 mol/dm3 at pH 4-8) or phosphate (0.05-0.1 mol/dm3 sodium or potassium salt at pH 2-8). In addition, polymeric reversed phase media also performs well at high pH and is often buffered with ammonium hydroxide or ammonium bicarbonate (0.05-0.1 mol/dm3 at pH 8-9). 

Bromophenylisocyanate (0.2 mmol) was combined with the Step 3 product in 1 ml toluene and heated to 100°C for 3 hours, then concentrated by centrifugal evaporation. The residue was dissolved in 5 ml DMF and purified by HPLC on a Cl8 silica gel column using acetonitrile/water gradients containing 0.05% trifluoroacetic acid as eluent. 

Diphenylchlorophosphate (0.3 mmol) was added dropwise to solution of benzoic acid (0.3 mmol) and triethylamine (0.3 mmol) dissolved in 1 ml dry CH2C12, then stirred 60 minutes at ambient temperature. This mixture was treated with the Step 5 product (0.3 mmol) and triethylamine (0.6 mmol) dissolved in 0.5 ml in dry CH2C12 and stirred overnight at ambient temperature. The solution was diluted with 2 ml CHC13, then washed with 2 ml 5M NaOH, and concentrated. The residue was dissolved in 5 ml methyl alcohol and purified by HPLC on a C18 silica gel column using acetonitrile/water gradients containing 0.05% trifluoroacetic acid as eluent and the product isolated. 

Most of the imprinted sorbents used in MISPE were prepared using MAA and ethylene glycol dimethacrylate (EDMA) as monomers. In these cases elution of more weakly bound analytes such as triazines [19,30,32], 7-hydroxy-coumarin (2) [26] or theophylline (12) [28] can be achieved using methanol or water as elution solvent. For more strongly bound analytes such as stronger nitrogen Bronsted bases, efficient elution has been achieved using eluents of the same base solvent but with the addition of small amounts of acids (e.g. acetic acid, trifluoroacetic acid (TEA)) or base (e.g. triethylamine (TEA)) [20-22,25,33]. 

Lincomycin in plasma (human or povine) and urine determined by HPLC at 214 nm using ion - pair HPLC column with an acetonitrile - trifluoroacetic acid ( 0.1 - 0.2 % v/v in the mixture) as an eluent (33). More than 1800 samples were assayed by this method. Soil - phase extraction (SPE) procedure was used in this analysis followed by an evaporation step. 

Figure 12.21 Separation of crude /5-MSH. (a) Analysis. Same column and temperature as in Figure 12.20 Linear gradient from 25% to 43% eluent B in A over 2.5 min eluent A 0.25% formic acid and 0.5% triethylamine in water eluent B acetonitrile flow rate 0.1 mL/min sample size 20 jig of crude /5-MSH. (b) Displacement. Same column and temperature as in Figure 12.20 carrier 0.1% trifluoroacetic acid in water displacer 50 mM BMDA in an aqueous solution containing 0.1% trifluoroacetic acid and 21% acetonitrile flow rate 0.1 mL/min fraction size 0.1 mL feed 30 mg of crude /1-MSH. Reproduced with permission from G. Viscomi, S. Lande and Cs. Horvdth, J. Chromatogr., 440 (1988) 157 (Figs. 4 and 5).

Sample preparation Prepare 500 mg 3 mL Bond Elut C2 SPE cartridges by rinsing with 2 mL MeOH then 2 mL 50 mM pH 7.5 TYis-HCl buffer. Apply 1 mL serum or CSF -I 1 mL 50 mM pH 7.5 Tris-HCl buffer to the cartridge and wash with 10 mL 50 mM pH 7.5 Tris-HCl buffer. Elute with 2 mL 50% MeCN containing 0.1% trifluoroacetic acid. Freeze dry eluent or diy an aliquot at 40° under a stretun of nitrogen, dissolve residue in 2560 p.L mobile phase, inject 20-200 p.L ahquot. 

The polymer was prepared similarly to the first example from a mixture of ethylene dimethacrylate (4.67 g), methyl methacrylate (0.22 g), A, A -diethyl-4-vinylbenzamidine (10a) (0.442 g 2.188 mmol) template 14 (0.350 g 1.094 mmol), AIBN (56.8 mg), and tetrahydrofuran (5.64 mL) as the porogen. Polymerization in a sealed ampoule was carried out for 70 h at 60 °C. After the usual workup procedure, the template was removed from the particles by extraction with methanol, followed by washing twice with 0.1 m NaOH/acetonitrile (1 1), water, and acetonitrile. The recovery of the template from the washings was determined by HPLC [RP8-column, eluent 0.2% trifluoroacetic acid in water/acetonitrile (55 45)]. Recovery of template was 85%. The inner surface area of the polymer amounted to 228 m g, and the swelling ratio in methanol was 1.45. 

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