Ionization suppression

Buffers are used in HPLC to control the ionization of one or more molecules in solution so that they will separate as sharp bands. The key to understanding ionization is to understand pH and pKa. 

The pH of a solution is simply a measure of the hydrogen ion concentration and represents the degree of harshness on either the acid or basic side. A pH of 7 is neutral and represents the mildest conditions. As we go toward lower pH, the hydrogen ion concentration increases and the solution becomes more harshly acidic. Starting at 7 and going toward higher pH, the hydrogen ion concentration decreases and the solution becomes a harsher base. 

Since buffers control pH best at their pKa, pick one close to your desired pH. The most common buffer used in HPLC is phosphate. It has two usable pKa s, 2.1 and 7.1, and is UV transparent. A 100-mM solution of phosphate precipitates in solution of 50% MeOH or 70% acetonitrile. Other buffers in common use are acetate, pKa 4.8, formate, pKa 3.8, and chloroacetate, pKa 2.9 all absorb in the UV below 225 nM. Sulfonate, pKa 1.8 and 6.9, should be substituted for phosphate when analyzing mixtures of organic phosphates. 

Amines pose an interesting problem for ionization control because their pKa are so high that they are usually ionized at any pH tolerated by the silica column bed. This makes them very soluble and hard to resolve on a reverse-phase column. It is possible to force them into the free amine form by using mobile phases at pH 12, but be sure to use a saturation column and change it often. 

Using the HPLC system with a mass spectrometer as a detector forces the use of volatile buffers to avoid contamination of the analyzer. The buffers are still needed in many cases to control sample or column ionization to improve the chromatography, but must be removed in some way before they reach the detector flow cell. A table of volatile buffers and their pKa s is listed in Appendix C. 

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More recently, liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) have been evaluated as possible alternative methods for carfentrazone-ethyl compounds in crop matrices. The LC/MS methods allow the chemical derivatization step for the acid metabolites to be avoided, reducing the analysis time. These new methods provide excellent sensitivity and method recovery for carfentrazone-ethyl. However, the final sample extracts, after being cleaned up extensively using three SPE cartridges, still exhibited ionization suppression due to the matrix background for the acid metabolites. Acceptable method recoveries (70-120%) of carfentrazone-ethyl metabolites have not yet been obtained. 

Online LC-ESI-TOF-MS experiments are carried out in a very similar fashion to the off-line NPS-HPLC separations described above, with a few notable exceptions. Firstly, 0.3% (v/v) formic acid is added to each mobile phase to counteract the ionization suppression induced by TFA. Because of the formic acid UV detection must be carried out at 280 nm (as opposed to 214 nm). To aid in normalization between runs 1 jag of Bovine insulin (MW = 5734 Da) is added to each chromatofocusing fraction prior to injection onto the column. Finally, the flow is split postcolumn directing 200 JlL/min into the ion source and the remaining 300 JlL/min through the UV detector and fraction collection. 

FIGURE 13.4 Total ion chromatograms from the ID LC/MS analysis of a yeast ribosomal protein fraction separated using 0.1% TFA (Panel a) and 0.1% formic acid (Panel b) as mobile phase modifiers. TFA produced narrower, more concentrated, peaks for mass analysis that did not overcome the significant electrospray ionization suppression associated with using this modifier for LC/MS studies, resulting in an overall reduction in component intensities. 

The peptides generated by proteolysis are separated using reverse-phase HPLC to minimize mass overlap and ionization suppression caused by ion competition in the electrospray source [40]. The optimized LC gradient parameters efficiently separate peptides while minimizing loss of deuterium through back exchange with solvent. Increased sensitivity can be achieved by using capillary HPLC columns and nanoelectrospray methods [47]. 

King, R. Bonfiglio, R. Femandez-Metzler, C. Miller-Stein, C. Olah, T. Mechanistic investigation of ionization suppression in electrospray ionization. J Am Soc Mass Spectrom 2000, 11, 942-950. 

Differing amounts of easily ionizable elements in real samples cause varying ionization suppression and hence the possibility of interference (see Section 2.4.2). 

Also called vapour-phase interferences or cation enhancement. In the air-acetylene flame, the intensity of rubidium absorption can be doubled by the addition of potassium. This is caused by ionization suppression (see Section 2.2.3), but if uncorrected will lead to substantial positive errors when the samples contain easily ionized elements and the standards do not. An example is when river water containing varying levels of sodium is to be analysed for a lithium tracer, and the standards, containing pure lithium chloride solutions, do not contain any ionization suppressor. 

Flame atomic absorption spectrometry can be used to determine trace levels of analyte in a wide range of sample types, with the proviso that the sample is first brought into solution. The methods described in Section 1.6 are all applicable to FAAS. Chemical interferences and ionization suppression cause the greatest problems, and steps must be taken to reduce these (e.g. the analysis of sea-water, refractory geological samples or metals). The analysis of oils and organic solvents is relatively easy since these samples actually provide fuel for the flame however, build-up of carbon in the burner slot must be avoided. Most biological samples can be analysed with ease provided that an appropriate digestion method is used which avoids analyte losses. 

Acetylsalicylic acid is readily absorbed from the gastrointestinal tract in dogs, cats, and swine due to its ionization suppression by the stomach acid. In the more alkaline small intestine, the large surface area for absorption makes up for the increased ionization of the drug and rapid absorption continues. In contrast, absorption is slower from the rumen in cattle. 

Liquid-liquid extraction is used extensively and successfully (6). If the analytes are acidic or basic, as is often the case when HPLC is the analytical method selected, appropriate ionization suppression can be employed to affect the desired extraction. Back extraction of the analytes into an appropriately buffered aqueous volume can then serve to isolate and concentrate. Anionic and cationic surfactants, or so-called ion-pairing reagents, can be added prior to extraction to increase the partition coefficients of the trace organic ionic compounds. 

An ionization suppressor decreases the extent of ionization of analyte. For example, in the analysis of potassium, it is recommended that solutions contain 1 000 ppm of CsCl, because cesium is more easily ionized than potassium. By producing a high concentration of electrons in the flame, ionization of Cs suppresses ionization of K. Ionization suppression is desirable in a low-temperature flame in which we want to observe neutral atoms. 

Just as there are recommended methods of analysis for the a- and /3-acids, so are there for the iso-a-acids (Table 2). Of these, two use tetraalkylammonium salt as an ion pair, whereas the other two rely on ionization suppression with phosphoric acid. Also evident is the variation in the de-... 

Acceptance. The RSD of the matrix factor for all lots must not deviate <15.0 %. If MF= 1, it indicates that there is no matrix effect. If MF >1, it indicates that there is ionization enhancement. If MF <1, it indicates that there is ionization suppression. 

Meng M, Bennett PK Source for imprecision resulting from ionization suppression from strongly retained phospholipids and dioctyl phthalate, Presented at 2004 ASMS conference, Nashville, TN... 

Barium. Before the routine use of AAS, Ba was analyzed by emission spectrograph or a KMnO spot test (13). Alkali and alkaline earth metals are analyzed in nitrous oxide/acetylene flames with ionization suppressants such as 1000 ppm Cs. For barium analysis by P CAM 173, background correction must be used whenever greater than 1000 ppm calcium is in the analyte solution. There are strong Ca(0H)2 absorptions and emission at 553.6 nm, which is the barium analytical line. 

King R, Bonfiglio R, Fernandez-Metzler C et al. (2000) Mechanistic Investigation of Ionization Suppression in Electrospray Ionization. Journal of the American Society for Mass Spectrometry 11 942-950... 

The most frequently chosen method for compound characterization in the pharmaceutical industry is LC/MS [6]. Replacing flow injection by a chromatographic separation prior to MS analysis offers three major advantages i) impurities or by-products are separated in time from the product of interest, rendering a purity assessment of the sample possible ii) ionization suppression of the compound of interest by salts, detergents, or by-products is avoided iii) the interpretation of mass spectra of pure compounds is much easier than the MS analysis of mixtures. Combinations of separation techniques with mass spectrometry have been reviewed recently by Tomer [39]. 

Although ionization of sodium is negligible and potassium small in an air—propane flame, some ionization is experienced in the recommended hotter air—acetylene flame. Ionization should be suppressed by the incorporation of excess potassium or cesium (for sodium determinations) or excess sodium or cesium (for potassium determinations), at concentrations of 1000/igml-1 or greater, in the form of chlorides or nitrates, in both sample and standard solutions. Cesium is the more effective but more expensive ionization suppressant. Extent of ionization is inversely related to analyte concentration with errors due to incomplete suppression thus being greater at low concentrations. As it is difficult to obtain alkali metal salts free from traces of other alkali metals, possible contamination must be considered, especially at low analyte levels. Use of a branched capillary for introduction of ionization buffer has been advocated for flame spectrometry to... 

Few elements interfere with the determination of sodium and potassium. Important interferences via ionization suppression from other alkali metals present in the sample are minimized as mentioned above. Interferences from high mineral acid concentrations on sodium and potassium absorption may be compensated for by matching sample and standard solutions with respect to acid type and concentration. For samples containing very high concentrations of sodium or potassium, the burner may be angled to reduce the need for excessive dilution. Alternatively the less sensitive (by a factor of about 150 for sodium and 200 for potassium) 330.24/330.30 nm (Na) and 404.41 nm (K) absorption lines may be employed. 

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