A well-established technique for accurate characterization of macromolecules is triple or tetra detection GPC. Including advanced detectors such as light scattering, in combination with a concentration detector, not only solves the issues of traditional GPC column calibration but also provides absolute molecular weight determination. Moreover, adding a viscometer which measures intrinsic viscosity can calculate the degree of branching or reveal structural differences.
In other words, sophisticated detection with several detector types, enables acquiring much more accurate information to be from a single injection.
After deciding that conventional column calibration is inadequate for their GPC analysis, customers might ask the following the questions:
- Will an existing GPC or HPLC be compatible with adding advanced detectors?
- Does the order of detectors matter?
- How to connect all the detectors without affecting the sensitivity or chromatography?
In this article, several common multi-detector GPC configurations are investigated and a comparison is drawn between modular detectors and integrated detectors.
Configuring a Multi-Detector GPC
A modular detector setup (Figure 1A) provides the advantages of additional flexibility to move around detectors to multiple systems. However, the benefits of moving detectors around have consequences – i.e., added dead volume between detectors which is not controlled by temperature. Inter-detector tubing is minimized by the integrated detector setup (Figure 1B) by positioning all detectors in close proximity in a temperature-controlled module. By minimizing inter-detector volume, dispersion and band-broadening as the sample passes from the first detector to the last detector are reduced. Band-broadening causes decreased sensitivity and poorer resolution of the latter detectors. In a multi-detector GPC system, which has three or more detectors, maintaining the resolution of chromatography throughout the whole system is crucial to realize high-quality data with the maximum signal-to-noise.
Figure 1. Schematic of A) a modular triple detector GPC setup compared to B) an integrated triple detector module.
The majority of multi-detector GPC systems arrange all detectors in series to avoid splitting flow with parallel detectors and maximize sample concentration. Modular detectors can effortlessly be attached to existing traditional systems with refractive index or UV detectors, but this generally doesn’t allow all detectors to stay in series. As the sample concentration decreases, the sensitivity is inherently decreased by any flow splits between the detectors. For flow to be split between two detectors such as the viscometer and refractive index, it must be ensured that flow splitting is equal across detectors. There must be no changes in inner diameter of tubing as this will support flow in one direction and lead to a much lower signal for the latter detector.
A 1 mg/mL concentration of 105 kDa polystyrene standard with polydispersity of 1.03 was prepared and injected into a tetra detection GPC system to evaluate the differences between an integrated and modular design. The injection volume was set to 100 μL. Different multi-detector configurations were compared to assess signal response and band-broadening.
Modular versus Integrated Detectors
While performing this comparison, the order of detectors was fixed with all respective detectors in series: UV, light scattering, refractive index, and viscometer. Figure 2 evaluates the response of the viscometer and light scattering signals for a modular design against the integrated design. The shift in retention time originates from the varied lengths of tubing between the detectors. For the modular design, the shape is a little wider and also there is a slight decrease in the peak height. These differences result directly from increased inter-detector dead volume in the modular design. In this comparison, the total inter-detector volumes of the integrated system and the modular system are 340 μL and about 680 μL, respectively.
Figure 2. Comparison of a modular (purple) and integrated (red) multi-detector GPC system. The light scattering (top) and viscometer (bottom) signals are shown to visualize the effects of sample dilution by adding more inter-detector tubing in the modular design.
Series versus Parallel Detectors
One usual configuration for including sophisticated detection to an existing traditional GPC system (Figure 3) is by splitting flow after the columns (or first detector) and running separately into following detectors. This enables the viscometer and the RI detector to be endmost in their respective detector sequences. In both cases, they are thus protected from the additional back pressure exerted by later detectors, which can cause damage to the RI flow cell or the viscometer pressure transducers.
Figure 3. Typical schematic of flow splits added when attaching advanced detectors to an existing HPLC or conventional GPC system.
In Figure 4, the viscometer and light scattering performance for the split flow modular design was compared against an integrated multi-detector design. There was a considerable reduction in response in the light scattering detector with peak shape distortion from band broadening. The software can add band broadening corrections; however, it cannot help the diminished light scattering sensitivity. This specific sample has a very narrow distribution, fairly high dn/dc of 0.185, and a molecular weight of roughly 100 kDa.
Achieving quality data with samples of broader distributions, lower dn/dc, and lower molecular weight demands much higher sample concentrations. Furthermore, the viscometer signal for the modular design showed a ten-fold decrease in signal and displays a significant band-broadening from dilution. The sensitivity of a viscometer depends greatly on flow rate, and hence the reduction in flow rate that comes with the flow split further compounds the loss of sensitivity. In this comparison, the integrated system has a total inter-detector of just 340 μL, whereas the modular system has around 860 μL of dead volume.
Figure 4. Comparison of a modular split flow (purple) and integrated (red) multi-detector GPC system. The light scattering (top) and viscometer (bottom) signals are shown to visualize the effects of sample dilution by adding more inter-detector tubing and splitting flow between detectors.
In certain cases, the flow split between the advanced and the refractive index detectors can become unbalanced and prefer one direction over another. This would be the case if the inner diameter tubing of the existing conventional GPC system is different than the tubing of the viscometer or light scattering detector.
Figure 5 compares the split flow modular setup, where different diameters of tubing were used between the advanced and the refractive index detectors, with the integrated multi-detector GPC. In this comparison, while the modular system has roughly 2.3 mL of dead volume, the integrated system has a total inter-detector dead volume of just 340 µL. The light scattering peak is fairly distorted from band-broadening, rendering the viscometer unusable. For triple or tetra detection, this configuration would not be recommended.
Figure 5. Comparison of an imbalanced modular split flow (purple) and integrated (red) multi-detector GPC system. The light scattering (top) and viscometer (bottom) signals are shown to visualize the effects of sample dilution by adding more inter-detector tubing and splitting flow between detectors.
When selecting the most suitable multi-detector GPC system, it is important to consider the trade-offs when incorporating modular detectors to existing traditional GPC systems. A modular detector does offer some apparent flexibility, but an integrated multi-detector GPC system retains the resolution of the columns while offering the highest sensitivity of detection among all detectors, generating the most information at the highest quality. When a conventional system needs to be upgraded, the various options should be looked into to ensure that the major requirement is being met. This particularly holds for a viscometer detector as its sensitivity is greatly dependent on the detectors being kept in series.
This information has been sourced, reviewed and adapted from materials provided by Malvern Panalytical.
For more information on this source, please visit Malvern Panalytical.