There are many variables to consider when configuring an HPLC system. To help, here are the top five questions you need to answer.
1. SAMPLE INJECTION: DO YOU NEED MANUAL OR AUTOMATED SAMPLE INJECTION?
HPLC samples are injected into the mobile phase either manually or automatically. Manual sample injection is sufficient when there are only a handful of samples to process, and can be easily configured in most systems. If you anticipate working with larger numbers of samples, automated sample injection is necessary. A variety of sample injectors exist to accommodate different capacities.
2. SAMPLE HANDLING: WHICH INJECTION METHOD IS RIGHT FOR YOUR SAMPLE VOLUME?
Syringe pumps, configured with an appropriate volume sample loop, can be used when working with samples of a consistent volume to provide a smooth, pulse-free flow. A solvent system provides higher throughput by accommodating larger sample volumes, as well as a wider range of potential volumes. Solvent systems also provide the ability to select from multiple solvent reservoirs for rinses, dilutions, and reagent additions.
3. PUMPING SYSTEM: HOW MUCH FLOW DO YOUR COLUMNS NEED?
The column size and stationary phase parameters will dictate flow rate needs and pressure requirements, and aid in the selection of the pumping system. Pumps can generate flow rates ranging from 10 μL/min to 500 mL/min, offering support for HPLC columns from 1 mm to 100 mm. Binary pumping systems are well-suited for simple, high-pressure mixing. Most of them are stackable, to save on bench space. Manometric modules and high-pressure mixers allow pressure monitoring and active mixing for smooth gradient formation. Pump heads constructed for bio-compatible applications are also available.
4. DETECTION: WHAT TYPE OF DETECTOR DO YOU REQUIRE?
Ultraviolet-visible (UV/vis) detectors are commonly used for purification, providing basic detection of analytes. Diode Array Detectors capture full spectra data, and allow peak purity checks and spectral library creation.
Evaporative Light Scattering (ELS) detectors are ideal for sensitive samples that do not contain chromophores. Scattered light is collected and measured to produce a signal.
Mass spectrometry (MS) detectors allow fraction to be collected based on target mass and save time by eliminating the need for subsequent analytical verification. Conductivity detectors monitor gradient formation and are especially important for processing of oligonucleotides and biological samples.
5. FRACTION COLLECTION: WILL YOU BE COLLECTING FRACTIONS?
Fraction collection is necessary in both preparative and semi-preparative applications, and whenever samples are to be further analyzed. Picking an appropriate fraction collector will depend on the expected number and volume of samples. Fraction capacity can be increased by either modifying the rack set, changing the vessels used or alternating where the fractions are collected.
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There are many possible configurations for HPLC systems, potentially making set up complicated. This article will help you develop a basic understanding of the main components of an HPLC system. With this knowledge, you’ll be achieving high resolution and fast separation of samples with ease.
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