HPLC buffers, common

TABLE 2 Common HPLC Buffers and their Respective pK, and UV Cutoffs... 

Amino acids Parenterals HPLC MeOH-0.1 /VHC1 (15 2), dilute with H,0, sonicate, mix and filter o-Phthaladehyde cm, 1 ml/min C18 (71 25 4) buffer-136 mg/ml KH P04, pH 3.0 Gradient Fluorescence same procedure for tablets with hydrochlorothiazide [1. 8,46.140] review1 [1046], HPLC [1105] Common amino acids [1039]... 

Table 2.3. Common HPLC Buffers and Their Respective pKa and UV Cut-off...

Some Properties of Buffers Commonly Used on RP-HPLC... 

Since it is necessary to remove solvent from the product, the mobile phase buffer must be considered. Some popular reverse-phase HPLC buffers, such as phosphates or zwitterion organic buffers, are nonvolatile. They must be replaced by a volatile buffer such as formic acid or ammonium acetate. Otherwise, a desalting step must be added. Trifuouroacetic add is another common buffer. Although it is fairly volatile, it forms a salt with the basic product and therefore cannot be completely removed from the final product. 

The use of buffers is common and is critical enough to many separations to warrant a detailed discussion here. As mentioned above, HPLC buffers are commonly used to control solution hydronium ion, H+. The H" " concentration [H+] is represented by the pH scale where pH = - log[H+]. 

The pKj values of some commonly used HPLC buffers are shown in Table 1. The table is divided into two parts buffers based on acids, and buffers based on a base. Of course, this list can be expanded at will. The buffers selected here cover the entire pH range of interest to the chromatographer. All organic buffers as well as hydrogencarbonate and ammonium buffers are volatile and are therefore compatible with MS detection, provided that a suitable counterion is used. 

In reverse-phase chromatography, which is the more commonly encountered form of HPLC, the stationary phase is nonpolar and the mobile phase is polar. The most common nonpolar stationary phases use an organochlorosilane for which the R group is an -octyl (Cg) or -octyldecyl (Cig) hydrocarbon chain. Most reverse-phase separations are carried out using a buffered aqueous solution as a polar mobile phase. Because the silica substrate is subject to hydrolysis in basic solutions, the pH of the mobile phase must be less than 7.5. 

Injecting the Sample The mechanism by which samples are introduced in capillary electrophoresis is quite different from that used in GC or HPLC. Two types of injection are commonly used hydrodynamic injection and electrokinetic injection. In both cases the capillary tube is filled with buffer solution. One end of the capillary tube is placed in the destination reservoir, and the other is placed in the sample vial. 

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. 

Buffers are used mainly to control the pH and the acid-base equilibrium of the solute in the mobile phase. They can also be used to influence the retention times of ionizable compounds. The buffer capacity should be maximum and should be uniform in the pH range of 2-8 commonly used in HPLC. The buffers should be soluble, stable, and compatible with the detector employed, e.g., citrates are known to react with certain HPLC hardware components. 

Reverse phase HPLC describes methods that utilize a polar mobile phase in combination with a nonpolar stationary phase. As stated above, the nonpolar stationary phase structure is a bonded phase—a structure that is chemically bonded to the silica particles. Here, typical column names often have the carbon number designation indicating the length of a carbon chain to which the nonpolar nature is attributed. Typical designations are C8, C18 (or ODS, meaning octadecyl silane), etc. Common mobile phase liquids are water, methanol, acetonitrile (CH3CN), and acetic acid buffered solutions. 

Numerous CE separations have been published for synthetic colours, sweeteners and preservatives (Frazier et al., 2000a Sadecka and Polonsky, 2000 Frazier et al., 2000b). A rapid CZE separation with diode array detection for six common synthetic food dyes in beverages, jellies and symps was described by Perez-Urquiza and Beltran (2000). Kuo et al. (1998) separated eight colours within 10 minutes using a pH 9.5 borax-NaOH buffer containing 5 mM /3-cyclodextrin. This latter method was suitable for separation of synthetic food colours in ice-cream bars and fmit soda drinks with very limited sample preparation. However the procedure was not validated for quantitative analysis. A review of natural colours and pigments analysis was made by Watanabe and Terabe (2000). Da Costa et al. (2000) reviewed the analysis of anthocyanin colours by CE and HPLC but concluded that the latter technique is more robust and applicable to complex sample types. Caramel type IV in soft drinks was identified and quantified by CE (Royle et al., 1998). 

The use of surface-enhanced resonance Raman spectroscopy (SERRS) as an identification tool in TLC and HPLC has been investigated in detail. The chemical structures and common names of anionic dyes employed as model compounds are depicted in Fig. 3.88. RP-HPLC separations were performed in an ODS column (100 X 3 mm i.d. particla size 5 pm). The flow rate was 0.7 ml/min and dyes were detected at 500 nm. A heated nitrogen flow (200°C, 3 bar) was employed for spraying the effluent and for evaporating the solvent. Silica and alumina TLC plates were applied as deposition substrates they were moved at a speed of 2 mm/min. Solvents A and B were ammonium acetate-acetic acid buffer (pH = 4.7) containing 25 mM tributylammonium nitrate (TBAN03) and methanol, respectively. The baseline separation of anionic dyes is illustrated in Fig. 3.89. It was established that the limits of identification of the deposited dyes were 10 - 20 ng corresponding to the injected concentrations of 5 - 10 /ig/ml. It was further stated that the combined HPLC-(TLC)-SERRS technique makes possible the safe identification of anionic dyes [150],... 

Buffers are commonly used to control the pH of the mobile phase, which greatly affects the retention of acidic or basic analytes. Table 1 summarizes the common buffers for HPLC and their respective pK and UV... 

Typical UV Cutoff Wavelengths for Some Commonly Used HPLC Solvents and Buffer Components, Taken Primarily from Manufacturer Product Specifications... 

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