HPLC buffers

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

HPLC buffer (0 2Mphosphate, pH 7.0) Add 0 2MNa2HP04 to 0 2MNaH2P04 to obtain required pH. 

The helix peptide was synthesized on a Gly-thioester resin 103 using standard V°-Boc chemistry SPPS. 104105 The peptide was deprotected and simultaneously cleaved from the resin by treatment with HF plus 2% PhOMe, 0 °C, 1 h. The crude peptide was taken up in neat TFA, diluted with double deionized H20 to 0.5% TFA and lyophilized to remove residual PhOMe. The helix was purified by semipreparative RP-HPLC (buffer A 0.1% TFA in H20 buffer B 90% MeCN+10% buffer A gradient 0-67% buffer B over 60 minutes at 3 mL min-1) and characterized by IS-MS calcd, 1372.6 found, 1372.24 (average isotope composition). 

Reverse phase HPLC buffers Buffer A 0.1% trifluoroacetic acid. Buffer B Buffer A containing 90% acetonitrile. 

Fill HPLC Buffer Reservoir A with PicoTag Eluant A and Buffer Reservoir B with 60% (vol/vol) acetonitrile in HPLC grade water. 

Fig. 1 Comparison of the two standard chemicals of a-lac-talhumin and P-lactoglohulin and whey proteins after ultrafiltration (MWCO 30,000) hy RP-HPLC (buffer A/huffer B=70/ 30-35/65 vol.%, gradient time of 15 min).

Prepare, filter, and degas HPLC buffers buffer A, H20 with 0.1% TFA buffer B, 80 20 (v/v) CH30H-H20 containing 0.1% TFA. Use a reversed-phase Qg column with detection at 214-220 nm. 

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

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. 

HPLC buffer A 20 mM ammonium acetate, pH 6.5 in water HPLC buffer B 20 mM ammonium acetate, pH 6.5, 70% acetonitrile... 

Equilibrate the SPE column(s) (1 ml HPLC buffer A, followed by 1 ml HPLC buffer B, followed by 3 ml HPLC buffer A). 

Analyze 50 pi of the dissolved, filtered sulfation reaction using the analytical RP-HPLC column with a Hnear gradient of HPLC buffer B from 5% to 60% over 40 min. If using an HPLC with a detector limited to one wavelength, monitor at 220 nm. 

Cation exchange wash 100 mMTris, 50 mM sodium chloride, pH 8.0 Cation exchange elution 100 mM Tris, 2 M sodium chloride, pH 8.0 RP-HPLC buffer A Aqueous 0.1% trifluoroacetic acid RP-HPLC buffer B Aqueous 0.1% trifluoroacetic acid, 70% acetonitrile RP-HPLC column 218TP510... 

Table 1 Reverse Phase HPLC Gradient Scheme Time (min) % RP-HPLC Buffer A...

RP-HPLC buffer A RP-HPLC buffer B Aqueous 0.1% trifluoroacetic acid. Aqueous 0.1% trifluoroacetic acid, 70% acetonitrile. ... 

Table 2 HPLC buffers used for the analysis of creatine and creatinine...

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. 

Which buffers are preferred for an application depends primarily on the choice of detector. For this reason, we distinguish between classical HPLC buffers, which are used with UV detection, and MS-compatible buffers, which are highly volatile. We discuss first the classical buffers. 

Besides the addition of add or base after the separation column, many analytes are converted to cations or anions by the formation of adducts. For example, compounds bearing OH functional groups tend to form adducts with alkali metal cations. The recommended alkali concentration is below 1 mmol L (i.e., soditun acetate) in order to avoid ion suppression effects, ft is important to achieve the cation adduct formation by post-column addition and under no circrunstances by addition to the HPLC buffer. This would cause a long-lasting contamination of the HPLC system. Carbohydrates can be detected as chloride adducts in negative-ion electrospray mode by adding 50 mM HCl (2 mmol L to the ESI ion source). 

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