Acidic modifiers/buffers acetic/formic acids

To achieve the optimum reversed-phase LC separation, one needs to explore variables such as the analyte chemistry, mobile-phase composition (solvent type, solvent composition, pH, and additives), column composition, column particle size, and column temperature. For pharmaceutical analysis using mass spectrometry, the chemistry of an analyte is rarely changed beyond manipulation of the mobile phase pH, and even there options are limited. Volatile pH modifiers (buffers) are still preferred for LC-MS, and concentrations of these modifiers are kept low. Relatively simply mobile phases consisting of water, acetonitrile, and either formic acid (0.1% v/v), ammonium acetate (1-20 mM), or both have been common. 

While earlier papers cited buffer systems or aqueous o-phosphoric acid to achieve satisfactory peak resolution, most recent investigations involved acetic acid or formic acid systems. " Representative examples are 0.2% and 1% HCOOH for betacyanins and betaxanthins, respectively, the latter requiring a lower pH for chromatographic resolution. Methanol or acetonitrile are most commonly used as modifiers, either undiluted or diluted with purified water at ratios of 60 40 or 80 20 (v/v), respectively. - Typical HPLC fingerprints for yellow and red beet juice are shown in Figure 6.4.1. 

Some commonly used buffers, such as sodium and potassium phosphate, are incompatible with ELSD, but there are ready alternatives. For example, ammonium acetate has similar buffering properties to potassium phosphate, and ammonium carbonate, ammonium formate, pyridinium acetate, and pyridinium formate are options for different pH ranges. Typical mobile phase modifiers that do not meet the volatility criteria can be replaced by a wide variety of more volatile alternates. For example, phosphoric acid, commonly used as an acid modifier fo control pH and ionization, can be replaced by trifluoroacetic acid other acids that are sufficiently volatile for use with FLSD include, acetic, carbonic, and formic acids. Triethylamine, commonly used as a base modifier, is compatible with FLSD other base modifiers that can be used are ethylamine, methylamine, and ammonium hydroxide [78]. 

Volatile buffers were reconsidered for the modified method. Triethylamine was ruled out primarily because it could not be obtained in high purity and because the secondary and primary amines contaminating it could potentially react with solutes present in the water sample. Preliminary evidence of reaction between ethidium bromide and triethylammonium bicarbonate was obtained, but the reaction product was not characterized. The components of volatile buffers that appeared acceptable on the basis of chemical purity were ammonia, acetic acid, and formic acid. A few exploratory experiments were conducted involving the elution by ammonium formate and ammonium acetate of EB or quinaldic acid exchanged onto AG MP-50 or IRA 900. These experiments showed that 1 M ammonium formate in water was a very poor eluent, but that EB could be eluted from AG MP-50 with 1 M ammonium formate in methanol. Elution was essentially complete with 6 bed volumes of the methanolic eluent, whereas neither methanol alone nor aqueous 1 M ammonium formate was able to elute this solute. This situation pointed out the necessity for a counterion to displace exchanged solutes and, additionally, indicated that the displaced solute be highly soluble in the eluting solvent. 

LC-MS methods are limited to mobile phase and column combinations that are compatible with electrospray ion sources. The mobile phase must be volatile and this usually means a combination of water acetonitrile methanol. The range of buffers and modifiers is also limited to volatiles, such as formic and acetic acid, or ammonium salts. Although most electrospray sources can accept flow rates of 1.0 mL/min, a flow rate of 0.2 mL/min or less is preferred therefore, LCMS methods for OA group toxins are usually reverse phase methods using 2 mm ID columns. Literature reports for the HPLC separation of OA group toxins before LC-MS include the following ... 

Lyophilize the purified end-labeled DNA that contains chloroacetaldehyde-modified MAR sequences at equal quantity in two separate Eppendorf tubes one for the hydrazine reaction and the other for the formic acid reaction. The step-by-step procedures for hydrazine and formic acid reactions for Maxam-Gilbert reactions are described in Sambrook et al. (1989). We have made the following deviations (a) the temperature employed for chemical reactions is 15°C instead of 20°C (b) at each step of precipitation of DNA with ethanol, there is no need to chill at -70°C before centrifugation and DNA is centrifuged at 10,000 g for 10 min at 4°C after the piperidine reaction, the sample is transferred to a new tube that contains 100 fi of 0.6 M sodium acetate at pH 5 in TE (10 mAf Tris-HCl, pH 7.5, 1 mAf EDTA) and precipitated with three volumes of ethanol. Redissolve the DNA pellet in 200 fi of 0.3 M sodium acetate and reprecipitate with ethanol. Wash the DNA pellet once with 70% ethanol, and lyophilize. Resuspend the DNA samples in 90% formamide in 1 x TBE (89 mAf Tris-borate, 89 mAf boric acid, 2 mAf EDTA) loading buffer, heat at 95 C for 5 min followed by quick chilling on ice. Separate the DNA samples on a polyacrylamide gel in 8.3 M urea, 100 mAf Tris-borate, pH 8.3, and 2 mAf EDTA. For best visualization of approximately 100-200 base pairs from the labeled end, 6% polyacrylamide gel is recommended. For visualizing 30-100 base pairs, an 8-10% polyacrylamide gel is typically used. 

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