Sodium acetate mobile phases

Type I CSPs have also been used with aqueous mobile phases. Pirkle et al. (32) have reported on the resolution of N-(3,5-dinitrobenzoyl) derivatives of M-amino adds and 2-aminophosphonic adds on an (l )-N-(2-naphthyl)-alanine-derived CSP using a mobile phase composed of methanol-aqueous phosphate buffer. The utility of achiral alkyltrimethylammoruum ion-pairing reagents was also investigated. Other examples include the following (1) The recently commercialized ot-Burke 1 CSP resolves the enantiomers of a number of underivatized p-blockers using an ethanol-dichlorornethane-ammonium acetate mobile phase (33) (2) an (R)-l-naphthylethylurea CSP was used to resolve N-(3,5-dinitrobenzoyI)-substituted amino adds and 3,5-dinitrobenzoyl amide derivatives of ibuprofen, naproxen, and fenoprofen with acetonitrile-sodium acetate mobile phases (34). 

Three phenylpropanoid glucosides (coniferin, kalopanaxin, syringin) were used to identify subspecies of Viscum album. These were extracted from powdered leaves and stems and separated on a C]g column (A = 264 nm). Baseline resolution and complete elution were achieved in 25 min using a 73.5/20/6.5 water/methanol/ water (0.1 M sodium acetate) mobile phase [397]. Calibration curves fi m 300 to 750ng/mL were used. 

Another application utilizing the coulometric array detector is the simultaneous determination of biogenic amines, kynurenine, and indole derivatives of tryptophan. The method employed a CIS column with a phosphate-acetate mobile phase (pH 4.1) containing methanol and sodium octyl sulphonate (Vaarman et al., 2002). 

Pd(II), Rh(III), Ru(III), and Pt(II) were baseline resolved as their 4-(5-nitro-2-pyridylazo)resorcinol complexes using a C g column (A = 536nm) and a 50/10/40 methanol/ethyl acetate/water (lOmM acetate buffer at pH 4.0 with lOmM tetra-butylammonium bromide and 0.01 mM sodium EDTA) mobile phase [155]. Separation was complete in 20 min and detection limits of - 1 ng/mL were reported. A plot of k versus percent methanol ranging from 40% to 90% with no ethyl acetate present was given. The k for some complexes exceeded 9 when the mobile phase contained <50% methanol, so this is probably not an effective system to use. Ethyl acetate was used to enhance the selectivity of the separation. Levels of 4% to 20% ethyl acetate were used to generate baseline separation. 

Sulfaguanidine, sulfadiazine, and succinyl sulfathiazole from a pharmaceutical powder were baseline resolved on a C,g column (A = 270nm) using a 20/80 methanol/water (50 mM ammonium acetate) mobile phase [508]. Elution was complete in 5 min and peak shapes were excellent. Twelve sulfonamides were well resolved on a C,g colunm (A = 254 nm) using an isocratic 6/94 IPA/water (20 mM sodium dodecylsullate [SDS] with phosphate buffer at pH 3.0) mobile phase. Elution was complete in 15 min. The effects of changing % IPA and SDS concentration on Id and a values were presented [509]. This study provides excellent background information for method development. Detection limits of 1 pg/mL were reported. 

The separation of Rh(lII), Ru(II), Pd(II), and Pt(II) as their 4-(5-nitro-2-pyridy-lazo)resorcinol complexes was achieved on a C g column (A = 536nm) using a 50/10/40 methanol/ethyl acetate/water (lOmM acetic acid buffer pH 4.0 with lOmM tetrabutylammonium bromide and lOmM sodium EDTA) mobile phase [155]. Elution was complete in <20 min. Peaks were baseline resolved. The detection limits were reported to be between 0.5 and 2.6ng/mL (ion dependent) and linear working concentrations were generated from 2 to lOOng/mL for all analytes. The authors note that ethyl acetate levels between 4% and 20% were effective for this separation but do not state that ethyl acetate is responsible for the selectivity obtained. 

Four organomercuiy compounds (medioxyetl l, ethyl, phenyl, and methyl) were extracted from water and separated from inorganic mercury (Hg " ") as their pyrrolidinethiocarbamate (PDTC) complexes [928]. A C g column (A not given) and a 60/40 acetonitrile/water (5mM sodium PDTC to pH 5.5 with ammonium acetate) mobile phase resolved these compounds in <10 min. Detection limits of 5 ng were reported. 

In a wonderfully short and effective separation, seven water-soluble vitamins (ascorbic acid, niacin, niacinamide, pyridoxine, folic acid, thiamine, riboflavin) were baseline resolved on a Cg column using an isocratic 7/93 acetonitrile/water (1% acetic acid and 5mM sodium heptanesulfonate) mobile phase [1114]. Elution was complete in 6 min. 

Cationic samples can be adsorbed on the resin by electrostatic interaction. If the polymer is strongly cationic, a fairly high salt concentration is required to prevent ionic interactions. Figure 4.18 demonstrates the effect of increasing sodium nitrate concentration on peak shapes for a cationic polymer, DEAE-dextran. A mobile phase of 0.5 M acetic acid with 0.3 M Na2S04 can also be used. 

FIGURE 10.13 Analysis of brome mosaic virus BMV on SynChropak GPC 500. Column 250 X 4.6 mm i.d. Mobile phase 0.05 A1 Bis-Tris, 0.5 A1 sodium acetate, pH 5.9. Flow rate 0.3 ml/min. (Reprinted from Jerson Silva and MICRA Scientific, Inc., with permission.)... 

For most free amino acids and small peptides, a mixture of alcohol with water is a typical mobile phase composition in the reversed-phase mode for glycopeptide CSPs. For some bifunctional amino acids and most other compounds, however, aqueous buffer is usually necessary to enhance resolution. The types of buffers dictate the retention, efficiency and - to a lesser effect - selectivity of analytes. Tri-ethylammonium acetate and ammonium nitrate are the most effective buffer systems, while sodium citrate is also effective for the separation of profens on vancomycin CSP, and ammonium acetate is the most appropriate for LC/MS applications. 

Fig. 7-9. Separation of amino acids after derivatization 5 with OPA and mercaptoethanol. Column Superspher 100 RP-18 (4 pm) LiChroCART 250-4, mobile phase 50 mM sodium acetate buffer pH 7.0/methanol, flowrate 1.0 ml min temperature 40 °C detection fluorescence, excitation 340 nm/emission 445 nm. Sample amino acid standard sample (Merck KGaA Application note W219180).

Mobile phase Methanol — water — dioxane — sodium acetate solution (aqueous, 0.2 mol/L, pH 8.0) (60-t-28-1-12+ 10)... 

Note The sodium acetate was added to the mobile phase solely to improve the separation. It had no detectable effect on the production of fluorescence during thermal activation, since the fluorescence reaction also occurred in the absence of sodium acetate. 

Chromatographic characterisation of hydrolysis products Hydrolysis products from sodium polypectate were analysed by thin-layer chromatography on silica gel G-60, using ethyl acetate / acetic acid / formic acid / water (9 3 1 4, by volume) as the mobile phase system. Sugars were detected with 0,2% orcinol in sulphuric add-methanol (10 90ml) [14]. 

Mobile phase The HPLC mobile phase is made up as follows. Prepare 2 L of acetate buffer by dissolving 13.6 g of sodium acetate and 6 mL of glacial acetic acid in 2 L of deionized water. Adjust the solution to pH 4.8 with concentrated sodium hydroxide solution (or glacial acetic acid) if necessary. Mix 2 L of buffer with 1.6-2 L (the amount depends on the particular commodity) of methanol. Eilter the solution through a 0.22-pm Nylon 66 filter membrane before using the mobile phase Absolute ethanol Aaper Alcohol and Chemical Co. (200 proof)... 

High-performance liquid chromatography (HPLC) with a micellar mobile phase or with a selective pre-column or reaction detection system has also been used to determine alkylenebis(dithiocarbamaes). ° Zineb and mancozeb residues in feed were determined by ion-pair HPLC with ultraviolet (UV) detection at 272 nm. These compounds were converted to water-soluble sodium salts with ethylenediaminetetra-acetic acid (EDTA) and sodium hydroxide. The extracts were ion-pair methylated with tetrabuthylammonium hydrogensulfate (ion-pair reagent) in a chloroform-hexane solvent mixture at pH 6.5-8.S. The use of an electrochemical detector has also been reported. ... 

Figure 4.23 Molecular weight distribution of a carboxynethyl-cellulose sasple. Coluiui combination of LiChrospher Si-100 and Si-500 mobile phase 0.5 H aqueous sodium acetate, pH 6, and flow rate 0.5 ml/min.

For non-aqueous mobile phases or mobile phases containing a high percentage organic modifier, ammonium perchlorate (0.05 M) or sodium acetate (0.05 M) may be added as supporting electrolyte. 

FMOC-amino acids can be chromatographed using a C8 column and acetonitrile in sodium acetate buffer as the mobile phase. Fluorescence detection with excitation at 260nm and emission at 31 Onm gives the best results. 

Most HPLC applications involving biomolecules utilize aqueous mobile phases. Critical parameters include both ionic strength and pH. Common solutes include TRIS, sodium phosphate, sodium acetate, and sodium chloride. Slightly alkaline pHs are preferable, for stability reasons. Specific examples of mobile phases include 50 mM TRIS, 25 mM KC1, and 5 mM MgCl2 (pH 7.2) for nucleotides, and 50 mM NaH2P04 (pH 7.0) and 20 mMTRIS and 0.1 M sodium acetate (pH 7.5) for both peptides and amino acids. All of these mobile phases are suitable for reverse phase or ion exchange applications. 

In a similar study with ESI the influence of different buffers was studied [12]. In the presence of acetic acid (HAc) only in the MeOH/H20 mobile-phase, a mass spectrum resulted with ion adducts of Na and K appearing as the most abundant ones. However, minor peaks could also be observed in the mass spectrum resulting from ammonium adducts (Fig. 4.3.2(A)). The respective ions could be suppressed or enhanced by changing the nature of the buffer used in the mobile-phase. For example, when a potassium buffer was used, sodium and ammonium adducts were suppressed, and the spectrum became less complicated with primarily the potassium adduct ion being visible (Fig. 4.3.2(C)). In addition, the signal-to-noise ratio improved by about a factor of 1.5-2. Similarly, sodium or ammonium acetate buffers enhanced the sodium and ammonium adduct ions, meanwhile suppressing other adducts (Fig. 4.3.2(B) and (D), respectively). 

Another study employed an ODS column and different mobile phase composition for the measurement of carotenoids in orange juice. Citrus fruits were hand-squeezed and the juice was filtered. Aliquots of 5 ml of juice were extracted with ethyl acetate (3 X 50 ml) containing 0.004 per cent butyl hydroxytoluene (BHT). The organic phase was dried with 50 g of anhydrous sodium sulphate and the aqueous phase was mixed with 50 ml of mehanol and 100 ml of 1 M NaCl, extracted with 75 and 25 ml of ethyl acetate. The ethyl acetate fractions were combined, evaporated to dryness at 40°C and redissolved in the mobile phase. Extracts were analysed in an ODS column (250 X 4.6 mm i.d. particle size 5 jian). The mobile phase consisted of ACN-methanol-l,2-dichloroethane (60 35 5, v/v) containing 0.1 per cent BHT, 0.1 per cent triethylamine and 0.05 M of ammonium acetate. The column was not thermostated and the flow rate was 1 ml/min. Pigments were detected... 

Various liquid chromatographic techniques have been frequently employed for the purification of commercial dyes for theoretical studies or for the exact determination of their toxicity and environmental pollution capacity. Thus, several sulphonated azo dyes were purified by using reversed-phase preparative HPLC. The chemical strctures, colour index names and numbers, and molecular masses of the sulphonated azo dyes included in the experiments are listed in Fig. 3.114. In order to determine the non-sulphonated azo dyes impurities, commercial dye samples were extracted with hexane, chloroform and ethyl acetate. Colourization of the organic phase indicated impurities. TLC carried out on silica and ODS stationary phases was also applied to control impurities. Mobile phases were composed of methanol, chloroform, acetone, ACN, 2-propanol, water and 0.1 M sodium sulphate depending on the type of stationary phase. Two ODS columns were employed for the analytical separation of dyes. The parameters of the columns were 150 X 3.9 mm i.d. particle size 4 /jm and 250 X 4.6 mm i.d. particle size 5 //m. Mobile phases consisted of methanol and 0.05 M aqueous ammonium acetate in various volume ratios. The flow rate was 0.9 ml/min and dyes were detected at 254 nm. Preparative separations were carried out in an ODS column (250 X 21.2 mm i.d.) using a flow rate of 13.5 ml/min. The composition of the mobile phases employed for the analytical and preparative separation of dyes is compiled in Table 3.33. 

FIGURE 1.25 HPLC determination of impurities in a levothyroxin (L-T4) formulation. Experimental conditions Column, Chiralpak QN-AX (150 rum x 4 rum ID) mobile phase, acetonitrile-50 mM ammonium acetate (60 40, v/v) (pHa 4.5) flow rate, 0.7 mLmiu UV detection, 240 nm temperature, 25 C. Sample, T4-200 tablets (Uni-Pharma, Greece) containing 0.2 mg L-T4 sodium per tablet the tablet was pulverized, suspended in methanol-10 mM sodium hydroxide (1 1 v/v) and after ultrasonication for 5 min the residues were removed by filtration. An aliquot of 10 xL of the filtrate was directly injected. (Reproduced from H. Gika et al., J. Chromatogr. B, 800 193 (2004). With permission.)... 

---