Microbore flow-rates and protein chromatography.

Reversed-phase chromatography of proteins on microbore columns can achieve sensitivities that exceed those for standard-bore columns by a factor of 10-20, when operated at the same linear velocities. These gains in sensitivity are accompanied by proportional reductions in peak volume. Sensitivities on standard- (4.6 mm I.D.) and narrow-bore (2.1 mm I.D.) columns have been further improved by reducing the flow-rates to those typical for microbore (1 mm I.D.) columns. We have investigated the role of flow-rate in determining peak volumes for a constant time gradient and found that flow-rate affects peak volume to a much greater extent than column diameter. Column length was not found to have a significant effect on either peak volume or sensitivity. We have found that a four-fold reduction in flow-rate results in at least a two-fold reduction in peak volume over the flow-rate range from 25 to 400 microliters/min. Recovery of proteins in smaller volumes should prove beneficial to subsequent protein characterization methodologies.

Postcolumn detection of serum proteins with the biuret and Lowry reactions.

The Lowry and biuret reactions have been adapted for the selective detection of chromatographically resolved proteins, specifically proteins separated by high-performance liquid chromatography. The protein reagents are continuously added to the column effluent and produce the characteristic chromophores with both proteins and peptides. The reaction chemistries are compatible with ion-exchange, steric exclusion, and reverse-phase chromatography. Detection limits for proteins resolved by ion-exchange are about 5 to 10 micrograms with the Lowry reaction. Peptides containing tyrosine can be detected at the 100-ng level when chromatographed on reverse-phase columns. The biuret reaction is about 8 times less sensitive for proteins and not very effective for peptides. Reaction detection can be combined with direct absorbance detection in the uv to distinguish proteinaceous peaks from other peaks containing uv-absorbing compounds.

Serum protein profiles by "high-performance" liquid chromatography with detection at multiple wavelengths.

We have separated serum proteins in only 15 min by high-performance liquid chromatography. The identification of several peaks in the chromatographic profile was greatly aided by the use of multiple-wavelength detection. We have found a good correlation between retention times and electrophoretic mobilities. Serum samples with increased gamma- or beta-globulins, as determined by electrophoresis, resulted in chromatographic profiles with strongly increased peaks in the appropriate regions. This chromatographic method revealed both relative and absolute differences in individual protein concentrations among several serum samples.

Interferences appearing in fluorometrically measured liquid-chromatographic profiles of creatine kinase isoenzymes in serum.

We observed nonenzymic peaks when serum isoenzymes of lactate dehydrogenase (EC 1.1.1.27; LD) and creatine kinase (EC 2.7.3.2.; CK) were separated by "high-performance" liquid chromatography and detected by continuously monitoring the column effluent for enzyme activity. Such background peaks were particularly apparent in CK isoenzyme profiles obtained from human sera. We observed two nonenzymic peaks with fluorescence detection, one in the CK-MB region, the other in the CK-BB region. Serum albumin was a major component in the artifactual CK-MB peak, with lipoprotein as a minor component. We present evidence that the material responsible for the other peak fluoresced quite strongly and is mostly pre-albumin.

New developments in analysis of isoenzymes separated by "high-performance" liquid chromatography.

We have developed two enzyme analyzers for use in "high-performance" liquid chromatography. In both systems two detectors are used, placed after the column effluent has been combined with assay reagent. In one system, an absorbance detector is placed before and after a post-column reaction coil. Peaks observed at one detector are subtracted from those at the other, to produce a two-point measurement of enzyme activity. The linear dynamic range was 17--1700 U/L for lactate dehydrogenase (EC 1.1.1.27). In the other system, two reaction coils were used and a single fluorescence detector was placed at the end of each coil. These coils were kept at different temperatures, and an automated switching valve diverted equal amounts of column effluent and reagent into both coils. The fluorescence readings were then subtracted to produce a differential measurement of enzyme activity. The linear dynamic range was 20--1000 U/L. We used both systems to chromatographically analyze lactate dehydrogenase isoenzymes, and could separately determine both the distribution and activity of sample isoenzymes.

Dual-detector-post-column reactor system for the detection of isoenzymes separated by high-performance liquid chromatography. I. Description and theory.

We describe a dual-detector-post-column chromatographic reaction detector system that corrects for substances present in biological samples that interfere with the measurement of isoenzymes separated on a chromatographic column. The response observed at the detector in front of the reaction coil is mathematically dispersed, time transformed and subtracted from the detector behind the coil to produce a blank corrected chromatogram. The same computer program calculates peak areas and other chromatographic parameters such as height equivalent to a theoretical plate and retention time. In addition, we have evaluated the dispersion effects caused by various changes in our experimental system.

Dual-detector-post-column reactor system for the detection of isoenzymes separated by high-performance liquid chromatography. II. Evaluation and application to lactate dehydrogenase isoenzymes.

We describe the separation of lactate dehydrogenase isoenzymes by high-performance liquid chromatography-anion-exchange columns and their quantitation by a computer-controlled, dual-detector post-column reaction system. The recoveries from the separation column were ca. 90%. The dynamic range of the system was linear over about three orders of magnitude from 3 to 1500 U/l. The coefficient of variation for isoenzyme peak areas was ca. 2%. The method is compared to the classical electrophoresis measurement and shows increased speed, resolution, precision and accuracy.

Techniques for detecting enzymes in high-performance liquid chromatography.

Techniques are described for the automated detection of a series of enzymes in a high-performance liquid chromatographic system. Detection was achieved by either a direct or a coupled enzyme assay using photometric detectors. In direct detection the immediate enzymatic product was monitored. Coupled enzyme assays required additional enzyme(s) to convert the product of the primary enzyme reaction into a more easily detectable form. The efficiency of both free and immobilized coupling enzyme(s) was evaluated. The detector sensitivity could be increased three-fold by increasing the reaction temperature. This system is particularly suitable for isoenzyme profiling in biological materials.

Rapid assessment of isoenzymes by high-performance liquid chromatography.

We describe the rapid profiling of isoenzymes by use of microparticulate anion-exchange chromatography supports and a continuous, post-separation enzyme detector in a high-performance liquid chromatograph. Chromatographic analysis and enzyme detection are fully automated and provide excellent reproducibility. Factors affecting the isoenzyme profile and detector response characteristics are assessed. Lactate dehydrogenase and creatine kinase isoenzymes in tissue extracts, control materials used as electrophoretic standards, and serum were profiled by this method to establish the resolution and reliability of the method. We show the clinical use of this method in detecting changes in these isoenzymes in serum associated with acute myocardial infarction.

A continuous-flow enzyme detector for liquid chromatography.

A detection system has been developed for the selective and sensitive detection of enzymes eluting from a liquid chromatographic column. This system monitors a reaction that the enzyme catalyzes and provides a chemical amplification ranging from 10(4) to 10(5). The detection system consists of a reagent or substrate pump, a post-column reactor packed with non-porous spherical glass beads, and a photometric detector. A linear and selective response to a series of enzymes of clinical importance is demonstrated.