“With multicomponent solutions, refractometry provides more of a fingerprint for quality control and final product checks,” says Alex White, product specialist at OptoTec (Ashland, VA). “But the technique is limited in what it can tell you about multicomponent solutions.”
Modularity to the rescue
Although refractometry is a mature technique, instrument developers continue to expand the method’s horizons in terms of utility and applications. Users obtain a lot more information by employing refractometry modularly in combination with add-on analyzers such as densitometers, polarimeters, turbidometers, colorimeters, viscometers, and titrators for pH and other determinations. “Modularity allows users to take multiparameter measurements on small sample volumes at high throughput,” White says. By connecting multiple instruments, users can quantify seven or more parameters and thus significantly upgrade the value of the analytical “fingerprint” for foods, pharmaceuticals, beverages, and other products. Automation— for example, plate handling and dispensing within the 96-well microplate format—further enhances throughput and walkaway time.
White calls systems that employ modularity “future proof.” The only restrictions are sample size and laboratory budgets.
RI as a detector
Another example of expanding refractometry horizons is the application of differential refractometry as a detection mode in ultrahigh-performance liquid chromatography (UHPLC).
UHPLC promises much faster analysis time, but that advantage comes at the cost of short column life and an intolerance for “dead volumes” outside the column. Dead volumes cause peaks to broaden, which flies against the whole point of UHPLC.
“Traditional refractometer detectors broaden peaks by twenty microliters or more,” says Michael Larkin, PhD, director of advanced projects at Wyatt Technology (Santa Barbara, CA). “That was acceptable when peaks were a hundred microliters wide or more, but with UHPLC’s twenty-microliter-wide peaks, with a conventional diffractometer you’ve just lost all your resolution.”
Wyatt’s release of a UHPLC-capable differential refractometer (DRI) with a three-microliter mixing volume has added refractometry to the ultrasensitive detection arsenal of modern chromatographers. “Before, UHPLC was practical only if your analytes possessed an ultraviolet chromophore,” Larkin says. “Now anything exhibiting a refractive index will do.”
Waters also sells a DRI suitable for UHPLC.
While UV is more sensitive than DRI, UV traces are not very useful unless the analyst knows the UV extinction coefficient, which is impossible for unknowns. Due to the unique way proteins and long peptides refract, the DRI response is almost completely independent of protein structure, while providing useful concentration data.
It comes down to a value known as dn/dc, a measure of protein refractive index as a function of concentration. “Amino acids have unique dn/dc values, but in proteins they fortuitously average, so every protein has about the same dn/dc value, plus or minus a few percent,” Larkin explains.
Customers value refractometry for its simplicity, says White. There are no moving parts, and instrumentation from all reputable vendors tends to be accurate and robust.
Differentiating characteristics include add-ons such as presses for solid samples and flow cells. Temperature control is essential, White says, because it influences the RI measurement. “Potential buyers should also put a premium on accuracy, stability, and traceability, whether they plan to use the instrument in stand-alone or modular mode.” Systems should have the capability of providing electronic signatures and a full audit trail as well as documenting who took measurements on what samples and any adjustments to the instrument configuration.
For additional resources on refractometers, including useful articles and a list of manufacturers, visit www.labmanager.com/refractometers