HPLC

After exploiting the chemical factors in the separation process, chromatographers can adjust column conditions – flow rate, column size, and particle diameter – to further improve separations.

This month's "Directions in Discovery" looks at column and mobile-phase selection as well as system components and modification. Making the right choices among these parameters will help analysts get the most out of their liquid chromatography–mass spectrometry systems.

This month's column begins our annual roundup of new column technologies shown at the Pittsburgh Conference. In part I, we'll look at HPLC columns and packings for reversed-, normal-, and bonded-phase; ion-exchange; ion; size-exclusion; and large- and preparative-scale chromatography as well as specialty columns.

In part III of this column series, John Dolan demonstrated that systematic variation of the solvent strength can change selectivity and retention, and changing solvent type can amplify the effect. This month, he looks at three additional parameters (pH, temperature, and column type) for adjusting selectivity.

The efficiencies of microbore-2 columns, which are prepared from blanks that have a wide variety of inner surface roughness, drop sharply when the size of individual surface roughness features approaches the particle size of the packing material. The results suggest that two categories of packed column structure relate to the surface features and yield high and low efficiency columns. This installment of "Column Watch" discusses this conclusion in terms of the stability of an agglomerated layer of packing particles on the blank wall when subjected to shear forces during column packing.

Adapting the organic solvent strength and type can be an effective way to improve an isocratic separation.

The authors investigated the two most popular types of interfaces for benchtop LC–MS systems – the particle interface and the atmospheric-pressure ionization interface – to learn how the information obtained separately from the two can be complementary.

In the second installment of this series, the author looks at the first phase of the life cycle of a new chromatography system, bringing together a number of elements of the workflow and the functions as they are used in the laboratory.

Guest author Paul Ross explains why porous graphite carbon may provide a solution to certain specific challenges in the retention and separation of very polar analytes and structurally similar compounds.

Most chromatographers can look at a chromatogram and provide a qualitative opinion about the separation, but it is equally important to be able to measure the separation quality.

Current sample preparation procedures for LC–MS as applied to samples from biological matrices.

LC problems fall into one of two categories: those associated with instrumentation and those associated with the separation itself. Dolan explains how to identify which kind you have and how to approach correcting the problem.

This report summarizes activities performed during and after World War II at Oak Ridge National Laboratory and Iowa State University aimed at the separation of rare earths by ion-exchange chromatography on laboratory, pilot-plant, and industrial scales.

More researchers are discovering that buying used laboratory equipment can be an effective way to reduce costs. An excellent supply of high-quality, used chromatography equipment is available on the market as a result of mergers and consolidations in biotechnology companies and downsizing in the environmental industry.

Monoliths are chromatography sorbents cast into columns as a single continuous piece in contrast with regular chromatographic sorbents, which are packed as individual particles. The guest authors compare three such novel sorbents with a conventional particle-packed column.

Displacement chromatography is a viable alternative to elution chromatography, but the biotechnology industry has yet to fully exploit this technique. The authors present results of a pilot-scale ion-exchange displacement process developed for the removal of variants from recombitant human brain-derived neutrotrophic factor.

Readers submit questions about how to isolate the source of carryover in LC methods, mobile-phase temperature effects, and the care of cleaning of columns.

Krull and Swartz examine validating cleaning methods for pharmaceutical manufacturing equipment and look at general requirements and specific cleaning procedures, sampling types, and analytical methods.

In this month's "Column Watch," Majors recounts some of the more interesting paper and poster presentations given at HPLC '99, held May 30–June 4, in Grenada, Spain.

Inadequate mobile-phase gassing may be the single largest cause of LC problems. Columnist Dolan examines bubble-problem sources and techniques that analysts can use to eliminate excess gas in the mobile phase.

The authors describe the results they've achieved by using water heated to 100–240 ºC as a liquid eluent for reversed-phase HPLC instead of an organic modifier. They point out that this alternative avoids many of the problems – toxicity, flammability, and cost – associated with organic modifiers.

The authors evaluate a new chiroptical detector's sensitivity for various chiral compounds, linear dynamic range, and relative response with different solvents.

A simple modification of the standard HPLC autosampler flow path enables automated sampling and analysis of samples outside the autosampler unit when users cannot place the sample container inside because of size restrictions.

Peak shape problems don't always have a single solution.

With the goal of developing an analytical method for the fast analysis of vitamins in a complex matrix, the authors created a method that used in-line and complementary HPLC with photodiode-array and MS detection techniques.