Concave gradient

The column used for blood serum analysis was 100 cm long, 1 mm in diameter and packed with RP 18 reversed phase having a particle size of 10 pm. A concave gradient program was used to develop the separation over a period of 45 min. at a flow rate of 50 pl/min. The initial solvent was 75% methanol 25% water and the final solvent was pure methanol. 

Ion-Exchange Increase salt concentration by 1.6 fold per t, in a concave gradient ... 

An HPLC assay, content uniformity and related compounds method for a blockbuster new drug product, was developed that utilized concave gradient elution, a flow rate of 1.25 ml/min and no temperature control of the column. Samples were placed in 1000-ml volumetric flasks, sample diluent was added, and the flasks were sonicated for 10 min followed by 30 min of mechanical shaking. This method was to be run in a QC laboratory at the contract manufacturing facility in Puerto Rico. While the developed method worked flawlessly in the development laboratory, the QC laboratory had many problems in performing the procedure. 

The polypeptide product was simultaneously removed from the resin and completely deprotected by treatment with anhydrous liquid HF. A mixture of 2.0 g of protected polypeptide resin and 2 mL of anisole (scavenger) in a Kel-F reaction vessel was treated with 20 mL of redistilled (from CoF3) anhydrous liquid HF at 0°C for 30 minutes. The HF was evaporated under vacuum and the residue of (pyro)-Glu-His-Trp-Ser-Tyr-3-(2-naphthyl)-D-alanyl-Leu-Arg-Pro-Gly-NH2,as its HF salt, was washed with ether. The residue was then extracted with glacial acetic acid. The acetic acid extract was lyophilized to yield 0.8 g of crude material. The crude polypeptide was loaded on a 4x40 cm. Amberlite XAD-4 column (polystyrene-4% divinylbenzene copolymer) and eluted with a concave gradient from water (0.5 L) to ethanol (1 L). The tubes containing fractions from effluent volume 690 mL to 1,470 mL were pooled and stripped to dryness to yield 490 mg of partially purified polypeptide. 

Eqn.(5.6) defines a so-called linear gradient. Indeed, linear gradients are most popular in RPLC [527]. In LSC, retention varies much more strongly with mobile phase composition than in RPLC, especially when small amounts of organic modifier are added to the mobile phase (see section 3.2.3). Therefore, concave gradients are to be preferred [527]. 

In eqns.(6.1) and (6.1a) a, b9 c and d are constants. Because

[Pg.263]

The pattern of the variation of retention with composition in LC is affected by the choice of both the stationary and the mobile phase. The optimum shape of the gradient for unknown wide range samples is dictated by the phase system. Linear or slightly convex gradients are optimal for RPLC. Concave gradients are optimal for LSC. 

The combination of these two factors determines the required shape of an LSS gradient. Linear gradients were shown to result for RPLC in section 5.4, whereas a concave gradient was found to be optimal for LSC in section 6.2.2. 

A strategy for the optimization of gradient programs based on the actual retention behaviour of some sample components has been described by Jandera and Chura5ek [623, 624]. This approach relies on the possibility to calculate retention and resolution under gradient conditions from known retention vs. composition relationships and plate numbers. Both typical RPLC (eqn.3.45) and LSC (eqn.3.74) relationships can be accommodated in the calculations and linear, convex and concave gradients are all possible because of the use of a flexible equation to describe the gradient function. This equation reads... 

To get a linear polarity gradient with the systems shown in Figure 9.2 would require a concave gradient. Choosing a gradient is often done by trial and error using an initial rate of 2% per minute. A variety of references are available for further details.42-44... 

HPLC Purification Steps. Reverse phase (R HPLC Step A Filtered samples were fractionated on a Supelcosil LC-18DB column with a Pelliguard guard column (Supelco) at ambient temperature on a Model 840 nquid chromatograph with autosampler (Waters). The sample was injected onto the column and eluted with a concave gradient Waters curve 7) over one hour at 1.0 ml/min beginning with 10% acetonitrile (0.1% v/v TFA) and 90% aqueous 0.1% IPA, and ending with 60% acetonitrile (0.1% v/v TFA and 40% aqueous 0.1% TFA. The eluant was monitored spectrophotometrically at 214 nm and by fluorescence (excitation = 230 nm, emission >300 nm). Fractions were collected over 1 min intervals, with both the autosampler and fraction collector cooled to 0-5 C. Fractions with the same retention times from multiple runs were pooled in the fraction collector and dried in the Speed-Vac concentrator ( . 500 BR-SOG/run). 

Figure 7-14. Elution of lactate dehydrogenase (LDH) isozymes with a concave gradient of NADH. Protein, 0.2 mg, in 0.2 ml O.IM sodium phosphate buffer, pH 7.0, ImM /3-mercaptoethanol, and IM NaCl was applied to an AMP analogue-sepharose column (140 X 6 mm, containing 2.5 g wet gel) equilibrated with O.lM sodium phosphate buffer, pH 7.5. The column was washed with 10 ml of the latter buffer, then the isozymes were eluted with a concave gradient of 0.0 to 0.5mM NADH in the same buffer, containing ImM /3-mercaptoethanoI. Fractions of 1 ml were collected at a rate of 3.4ml/hour. The hatched area indicates the pooled fractions that were rechromatographed. [From P. Brodelius and K. Mosbach, FEBS Lett, 35 223 (1973).]...

Finally, the two lowest chromatograms show the effect of the shape (curvature) of the gradient on separation. With a convex gradient shape (A) the earlier-eluted peaks are more bunched together than the later-eluted ones, whereas the opposite effect is observed with a concave gradient (B). 

Step 3. Decrease the initial conditions. This is called a concave gradient. 

Fig. 9.6. Composite diagram showing the elution position of some peptides and amino acids. Chromatographic conditions column, Lichrosorb Si 60 7 fim (treated with 0.1 M copper(II) sulphate/1 M ammonia) mobile phase, (A) water/acetonitrile (10 90)-0.1 M ammonia, 1 ppm Cu " ", (B) water-acetonitrile (60 40)-0.95 M ammonia, 1 ppm Cu " ". Elution was achieved with a concave gradient of 0% B to 100% B over 70 min flow rate, 2 ml/min detection, UV at 254 nm. 1, Phe-Phe 2, Ala-Ala-Ala 3, mixture 4, Ala-Ser 5, Pro-Glu 6, Phe 7, Gly-Gly-Gly 8, Lys-Phe 9, Leu 10, Leu 11, Glu 12, Ala 13, Ser-Ser-Ser 14, Gly-His-Gly 15, Arg-Glu 16, Lys-Gly 17, Arg-Tyr 18, Pro-Gly-Lys-Ala-Arg,Lys-Lys-Gly-Glu A, hydrophobic, large peptides B, dipetides C, amino acids, hydrophilic peptides, basic peptides.

Fig. 11.6.2. HPLC separation of steroid standards. Chromatographic conditions column, Hypersil ODS (150x4.6 mm O.D.) mobile phase, concave gradient (dashed line) of methanol-water (40-60% methanol) flow rate, 1 ml/min temperature, 45 °C detection, UV at 240 nm. Peaks A, 11-dehydrocorticosterone AD, androstenedione ALDO, aldosterone, B, corticosterone DHP, dihydroprogesterone DOC, 11-deoxycorticosterone E, cortisone F, cortisol G, adrenosterone, P, progesterone S, 11-deoxycortisol T, testosterone. Reproduced from O Hare and Nice (1982), with...

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