Analysis supports every stage of research and development, and for biomedicine that means high-performance liquid chromatography (HPLC) or liquid chromatography (LC). Although the process of selecting an HPLC system is unique for every circumstance and every life science laboratory, enough commonality exists to justify a 1000-word guide.
Commercial HPLC was introduced by Waters Associates (now Waters Corp.) in 1967, and by the 1980s was found in many (if not most) biology and pharmaceutical laboratories. The technique underwent a significant upgrade in 2004 with the debut of Ultra-Performance Liquid Chromatography (UPLC), a Waters trademark. Competitors were at first critical, mainly of the high pressures (and attendant issues) required to operate UPLC and its sub-2-micron columns, but eventually every major manufacturer developed their own versions, which came to be known generically as uHPLC.
UPLC or standard LC?
UPLC/uHPLC, says Jim Karafilidis, Principal Product Marketing Manager at Waters, led to dramatic increases in resolution, speed, and sensitivity in liquid chromatography, and raises interesting questions about matching systems to workflows.
“Analytical scientists consider a number of factors when choosing between HPLC or UPLC instrumentation. One important factor is purchase price, which at $30k to $65K is lower for HPLC systems than for UPLC, which is in the $55K to $120K range. Ongoing costs, which include maintenance, support, and consumables, are an additional concern. However, while the initial purchase price of a UPLC system is higher than that of HPLC system, lifetime operating costs are typically lower for UPLC, which requires much less sample and solvent than what is required for HPLC systems.”
Deciding between HPLC and UPLC, then, often comes down to productivity and efficiency. UPLC systems offer ultrafast analysis that cuts run time by a factor of ten and solvent consumption by fifteen-fold. “This allows scientists to process significantly more samples on UPLC systems in the same amount of time. UPLC systems also are more sensitive and offer greater resolution and higher peak capacity.”
Lastly and, Karafilidis suggests most importantly, scientists choose LC systems based on their unique methods and needs. That choice, he says, “is based on criteria outlined in methodology established by government entities such as AOAC International or various pharmacopeias, or internal standard operating procedures, which dictate the LC instrumentation and column technology that must be implemented to obtain compliant and scientifically valid results.”
Detectors
Photodiode arrays (PDA) and tunable ultraviolet (TUV) absorbance are the most common detector types used in the life sciences, particularly in quality laboratories. PDAs and TUVs detect chromophores —regions on molecules that absorb UV or visible light—and acquire UV-Vis spectra over the entire wavelength range, from 100 nm to 800 nm, or over selected ranges of wavelengths. TUVs may also be set to monitor up to four selectable wavelengths. “These detectors are best suited for small molecule pharmaceutical, natural product, or large molecule biopharmaceutical analysis,” Karafilidis tells Biocompare.
Fluorescence detection, or FLR, is the mode of choice for molecules containing fluorophores (as opposed to chromophores). FLR detectors are used for fluorophore-containing environmental or food samples, or for appropriately labeled proteins, amino acids, or glycans.
“If a sample lacks either chromophores or fluorophores, then a universal detector such as an evaporative light scattering detector (ELSD) may be the right choice,” he advises.
Another specialty detector, based on refractive index, is most often used to analyze sugars, polymers, surfactants, or other compounds that lack chromophores.
“Mass detectors are used when confirmatory or more-precise compound identification is required. Modern mass detectors are compact, rugged, built for routine analyses, and lessen the risk that a trace impurity in a sample will go undetected due to a coelution. The typical mass detector is highly reproducible and accurate over several orders of magnitude in analyte concentration. “
“Where called for, it is not unusual for analytical methods to feature two or even three detectors in series to fully characterize a sample.”
Fluid path composition
Another factor in HPLC selection involves the material composition of the fluid path. Buyers now have choices, including stainless steel, titanium, PEEK, or one of a new class of liquid chromatographs featuring novel surface technologies.
Since the earliest days of HPLC, stainless steel has been the standard material of construction for chromatographs and columns.
“Stainless steel is strong, readily available, and relatively inexpensive,” Karafilidis says. “However, it’s widely known that stainless steel hardware can cause poor peak shapes and recoveries for certain analytes containing phosphate and/or carboxylate groups, while other analytes with electron-rich functional groups are as difficult to measure accurately. Asymmetric peak shapes and poor recoveries for these analytes only worsen when stainless steel corrodes over time when exposed to highly acidic mobile phases or those containing chloride salts.”
For decades, the work-around was to use alternative metals and polymers, such as titanium and nickel-cobalt alloys or PEEK, for components in the flow path of HPLC systems.
“While these materials improve corrosion resistance, non-specific adsorption can still cause problems. Another temporary work-around approach is to add chelators like EDTA, citric acid, acetylacetone, or medronic acid to LC-MS mobile phases. However, chelators suppress ions and may be difficult to remove from HPLC systems. Chelators are also known to corrode stainless steel.”
The most recent iteration of UPLC and uHPLC systems eliminate these issues altogether by masking the active sites in the flow path of HPLC systems and columns, rendering them inaccessible to the analytes.
“Hybrid surface technology based on an ethylene-bridged siloxane polymer achieves this by forming an effective barrier that prevents the analyte molecules from coming in contact with metal surfaces of the instrument and analytical column,” Karafilidis says.