Amino Acid Analysis Part 2
HPLC System Performance Factors
If the desired separation quality and reproducibility can be achieved, then quality and reproducibility of the post-column derivatization will follow. However, this statement presupposes that the HPLC system used will meet all of the basic requirements for holding and injecting a sample, forming the eluant gradient or step-change, pumping eluant through the column against high pressure, and detecting the signal from the post-column reaction with low background noise and high sensitivity. For most HPLC brands these assumptions are valid.
Ion-exchange chromatography of amino acids followed by post-column derivatization places higher performance demands upon the HPLC system than do standard reversed-phase applications. Thus, for methods requiring an elution gradient, eluant pumps with low-pressure mixing are preferred. These are the exemplified by a ternary gradient pump with a proportioning valve which delivers a pre-programmed percentage of each eluant per pump stroke, in order to form the desired elution gradient.
Since all Pickering gradient methods require three eluants– two buffers and a high-pH column regenerant– a ternary HPLC pump is required. If a piston-wash accessory is available for that system, we strongly recommend its use, since it will prolong the life of the piston seal. The additional expense of a more inert pump and flow-path than stainless steel, e.g. titanium, while desirable, is not needed for these applications. The advances in HPLC technology over the last ten years have resulted in commercially-available HPLC gradient pumps which are capable of stable, highly-reproducible operation against high pressures.
Caution: Some HPLC pumps require special seals, check-valves or other components to be compatible with high pH or high-concentration salt buffers. Contact the pump manufacturer or Pickering Laboratories for guidance.
For special step-gradient applications such as PKU and all of the Beckman System 6300 methods, pumps delivering eluants via high-pressure mixing have an advantage over the HPLC ternary gradient systems. Since each pump delivers 100% of one eluant at a time, the “mixing” takes place after the pump outlet, before the column. Discrete step changes and return to equilibrium conditions can be sharp and rapidly effective, because the liquid in each pump head is not a mixture, and consequently, the volume does not need to be swept out beforehand. For the 5-10 minute high-volume clinical screening analyses, especially, it is highly desirable to be able to recycle the column as rapidly as possible for the next sample injection.
Depending upon the design and volume of the flow-path in the HPLC pump head, it may be necessary to “dead-head” the pressure transducer and/or capacitor. It is important to ensure that there is no “dead volume” where unswept buffers may reside and adversely affect the reproducibility of the early part of the chromatogram.
Before ordering a post-column amino acid analysis system, be sure to let us know what brand of HPLC equipment you intend to use. Our Customer Service department can advise you of the required modifications, if any.
Whether using a manual injector or autosampler, the injection valve in most brands (usually Rheodyne) contains a rotor seal which will not tolerate the high pH column regenerant used in the amino acid methods. Some, like Hewlett-Packard’s, also have an injection needle seat of the same material. Lithium, Potassium or Sodium Hydroxide will dissolve the standard seal, and the dissolved polymer will be distributed eventually throughout the column, post-column system, and the detector. Besides causing chronic baseline perturbations, such contamination may result in the near-term replacement of the column, guard, and reactor. This unfortunate outcome can be prevented by substituting the original rotor seal (usually Vespel®) with one made of either Tefzel® or PEEK.
While all-titanium construction is not necessary, a titanium sample needle will extend the service interval significantly.
Dip-tubes, gas dispersers and filters in the eluant reservoirs are subject to corrosion by the sodium and lithium buffers. All such metal parts in contact with these liquids must be replaced by inert equivalents.
In post-column amino acid analysis, the reagent and eluant stream meet at the mixing Tee, are heated, and then directed to the detector. If OPA is the post-column reagent of choice, most major brands of fluorescence detectors will work without modification– some more sensitively than others. The post-column instrument applies a nominal 7 bar (100 psi) to the flowcell outlet in order to prevent bubble formation, but this pressure can be adjusted downwards if the flowcell will not tolerate 7 bar.
For the visible-range detector required for the Ninhydrin reagent, the same pressure specification applies. In addition, the geometry of the flowcell should reject refractive index (RI) effects. Expect a very noisy baseline if the detector flowcell is not RI insensitive. Fortunately, most modern brands of detector flowcells are designed to reduce or eliminate the effects of RI.
For detection at 570 and 440 nm a detector with a tungsten lamp is recommended, but not strictly necessary. Background subtraction at 690 nm is a useful feature for improving the signal/noise ratio.
Some detector flow-cells have a very fine inlet capillary or a heat-exchanger. If they are too restrictive, the excess back-pressure on the post-column reactor will cause the safety relief valve to open. Such tubing can be safely replaced by 0.25 mm (0.010 inch) i.d. tubing. Consult Pickering or the manufacturer of the detector for specific information.