ADVANTAGES ANDDISADVANTAGES OF HPLC
The first things we need to understand are how an HPLC system works, itsbest applications and advantages over other ways of separating compounds,and other techniques that might compliment or even replace it. Is there afaster, easier, cheaper, or more sensitive method of achieving your results? Theanswer is yes, no, maybe. It really depends on what you are trying to achieve.HPLC’s virtue lies in its versatility! I have used it to separate compoundsof molecular weights from 54 to 450,000 Daltons. Amounts of material to bedetected can vary from picograms and nanograms (analytical scale) to microgramsand milligrams (semi-preparative scale) to multigrams (preparativescale). There are no requirements for volatile compounds or derivatives.Aqueous samples can be run directly after a simple filtration. Compounds witha wide polarity range can be analyzed in a single run. Thermally labile compoundscan be run. I had one customer whose company made explosives forprimers. Her first job of the day was to explode samples of the previous dayrun with a rifle. Her second job was to carry out an HPLC analysis of thatday’s run.An HPLC offers a combination of speed, reproducibility, and sensitivity.Typical runs take from 10 to 30 min, but long gradients might consume 1 to2 hrs. I have seen 15- to 30-sec stat runs on 3-mm columns in hospital laboratories.Retention times on the same column, run to run, should reproduce by1%. Two columns of the same type from the same manufacturer should give5% or better retention time reproduction on the same standard set.While the HPLC can be used in a variety of research and productionoperations, there are a few places where it really shines. Because it can rununderivatized mixtures, it is a great tool for separating and analyzing crudemixtures with minimum sample preparation. I began my HPLC career analyzingherbicide production runs as a method of trouble-shooting product yieldproblems. HPLC was routinely used in the quality control lab to fingerprintbatches of final product using a similar analysis. I have helped my customers runtissue extracts, agricultural run-off waters, urine, and blood samples withminimum clean up. These samples obviously are not very good for columnswhose performance degrades rapidly under these conditions. Columns canusually be restored with vigorous washing, but an ounce of prevention is generallymore effective than a pound of cure and also much more time effective.Standards purification is another role in which the HPLC excels. It is fairlyeasy to purify microgram to milligram quantities of standard compounds usingthe typical laboratory system.Finally, used correctly, HPLC is a great tool for rapid reaction monitoringeither in glassware or in large production kettles. I started my analytical careerwith a HPLC system cast-off by the Analytical Department and a 15-min trainingcourse by another plant monitoring chemist. He gave me an existing HPLCprocedure for my compound and turned me loose.The next day I was gettingresearch information. I could see starting material disappear, intermediatesform, and both final product and by-products appear. It was like having awindow on my reaction flask through which I could observe the chemistry ofthe ongoing synthesis. Later, I used the same technique to monitor a productionrun in a 6000-gallon reactor. The sampling technique was different, butthe HPLC analysis was identical.Versatility, however, brings with it challenge. An HPLC is easily assembledand easily run, but to achieve optimum separation, the operator needs tounderstand the system, its columns, and the chemistry of the compounds beingseparated. This will require a little work and a little thought, but the skillsrequired do offer a certain job security.I don’t want to leave you with the impression that I feel that HPLC is theperfect analytical system.The basic system is rather expensive compared withsome analytical tools; columns are expensive with a relatively short operatinglife, solvents are expensive and disposal of used solvent is becoming a realheadache. Other techniques offer more sensitivity of detection or improvedseparation for certain types of compounds (i.e., volatiles by GLC, largecharged molecules by electrophoresis). Nothing else that I know of, however,offers the laboratory the wide range of separating modes, the combination ofqualitative and quantitative separation, and the basic versatility of the HPLCsystem.