Gradient elution systems

An HPLC (39) method was developed for determination of the drug and its metabolites in human and rat bile. A stainless-steel column (15 cm X 4.6 mm I.D.) packed with UChrosorb RP-8 (Pore size 5pm) or Nucleasil Ci8 (pore size 5pm) was used. The columns were packed by means of a balanced density slurry method specially developed for the ammonia elution system. Gradient elution was performed with water (0.005 M ammonia) to which methanol was added, according to the desired programme. The final elution was usually effected with 100% methanol. Flow rate was Iml/min. A wavelength of 235 nm was found suitable for the detection of drug and its metabolites. 

DALLUGE J J, NELSON B c, THOMAS J B and SANDER L 0 (1998) Selection of column and gradient elution system for the separation of catechins in green tea using high-performance liquid chromatography , J Chromatogr A, 793, 265-74. 

The limit of detection for this instrument is about 10 pg/ ml for polystyrene in 2-butanone,163 which is close to two orders of magnitude higher than that of the deflection-type DRI. Moreover, the response of the ELSD is linear over only two decades in concentration.163 The ELSD is a useful backup detector when the DRI or UV detectors are not appropriate, e.g., when the UV absorbance or RI change is a function of copolymer composition as well as concentration or in gradient elution systems where changes in solvent composition cause drift in baselines of the UV and DRI detectors. Compounds about as volatile as the solvent are poorly detected by ELSD. 

If you use only one liquid, either neat or as a mixture, the entire chromatogram is said to be isochratic. There are units that can deliver varying solvent compositions over time. These are called gradient elution systems. 

The rise in popularity of HPLC is due in large part to its high-performance nature and the advantages offered over the older, noninstrumental open-column method described in Chapter 11. Separation and quantitation procedures that require hours and sometimes days with the open-column method can be completed in a matter of minutes, or even seconds, with HPLC. Modern column technology and gradient solvent elution systems, which will be described, have contributed significantly to this advantage in that extremely complex samples can be resolved with ease in a very short time. 

Another screening strategy using the same type of columns but with normal-phase gradient elution has been proposed by the pharmaceutical group Lilly.In this strategy, each compound is screened on four columns, i.e., Chiralcel OD-H, Chiralpak AD, Chiralpak AS and Chiralcel OJ. An n-hexane/2-propanol and an n-hexane/EtOH gradient elution system are... 

Dams et al. [19] determined seven different opium alkaloids and derivatives in seized heroin using fast LC-MS analysis. Analytes were separated in 5 min on a monolithic silica column with a gradient elution system and an optimized flow of 5 mL/min. Detection was carried out using a sonic spray ion source [20] a modified ESI source were ionization is achieved using a nebulizer gas at sonic speed instead of applying an electrical field. 

The sample extracts that show either toxicity or no dose response on initial testing should be fractionated. An aliquot of the extract is solvent exchanged to acetonitrile, and an initial analytical scale separation is made to assess the distribution of constituents in the sample. This separation is accomplished by using a Qg reversed-phase system eluted for 45 min with a linear gradient of 0-100% acetonitrile in water. If >75% of the sample elutes after the solvent composition of 80% and 20% acetonitrile, then the fractions are isolated by preparative reversed-phase HPLC. Fraction A is eluted with 100% water fraction B is eluted with a linear mobile-phase gradient from 100% to 75% water and 25% acetonitrile fractions C, D, and E are eluted with gradients with final compositions of 50%, 75%, and 100% acetonitrile. 

When performing HPLC (see Basic Protocol 2) the time of analysis will depend on the conditions used—i.e., isocratic versus gradient. Isocratic separation of the individual betacyanins, as shown in Figure F3.1.2, requires 20 min, compared to 9 min using a gradient elution system. 

In the beginning, the refractive index detector was the most used detection system, although it has two important drawbacks (1) solvent gradients cannot be used, and (2) it has low sensitivity and different responses to saturated and highly unsaturated TGs (112). Moreover, use of the ultraviolet (UV) detector is difficult, because the most adequate solvents also absorb in the same range and therefore cause an important baseline drift with gradient elution systems (106). 

Figure 10.9 HPLC trace of standard pigments using gradient elution system, (a) standard mixture (1) monitored at 415 nm (b) standard mixture (2) monitored at 490 nm and 590 nm. Conditions column 5 pm ( IS 150 X 4.6 mm, using diode array detection at 450 nm and gradient elution solvent A = 0.02 M ammonium acetate, Solvent B = acetonitrile gradient profile 0 min 95% A, 20 min 50% A, 25 min 95% A flow rate 1.0 ml/min.

The front-end processor also provides a visual warning flag when the reference array saturates. This is done by comparing the signal level received from the reference array with a manually set upper limit. Saturation of the reference array is not a problem when the detector system is run with a reverse phase or any single solvent system. The problem becomes very real when gradient elution systems are employed. 

Figure 2 (Opposite) RP HPLC chromatograms of the Alzheimer s Ap 1-42 peptide synthesized by different protocols for activation of the Fmoc-amino acids. Analytical RP HPLC employed a Waters system with a Vydac C4 (214TP54) column at a flow rate of 1.0 ml/min. The peptides were eluted by gradient (5-95 % B, 60 min) with 0.1 % TFA (buffer A) and 0.1 % TFA / acetonitrile (buffer B). a. Crude product obtained using BOP/HOBt/NMM 40 °C acylations + 40 °C deprotections, b. Cmde product obtained using BOP/HOBt/NMM 40 °C acylations 55 °Cdeprotections. c. Crude product obtained using Preformed Fmoc-aminoacyl fluorides 40 °C acylations 55 C deprotections, d. RP HPLC purified peptide.

Miyazaki et al. (1966, 1967 Miyazaki and Takemura 1966) have used column chromatography on DEAE-Sephadex A-25 to fractionate tRNA from Torulopsis utilis. They were able to purify an alanine, a valine and an isoleucine tRNA using 3 elution systems 1) Linear gradient of ammonium sulphate in 0.02 M potassium acetate, 1% dimethylformamide, pH 5.3 2) Linear gradient of KCl in 1 M potassium phosphate + 5% dimethylformamide, pH 6.1 or 6.2 at 30°C ... 

However, if two nonidentical chromatographic modes with some degree of similarity are used in a 2D system, the increase in the PC and the total number of analytes that can be separated is much lower than the product of peak capacities of individual dimensions. The PC also depends on the elution mode. Gradient elution provides a higher PC than isocratic elution and is of advantage in 2D LC. 

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