The sample management system in the Waters 2695 Separation Module uses five carousels with total capacity of 120 vials. A carrier rotates the carousels to the injection station in the sample compartment. Our GPC is employing the two primary detectors: 2414 Refractive Index Detector and 2996 Photodiode Array (PDA) Detector.

Refractive index is used for compounds that do not have strong UV chromophores, fluorophores, electrochemical, or ionic activities. Traditionally, refractive index detectors have been used for carbohydrates and lipids. The refractive index is used in polymer analysis by gel permeation or size exclusion chromatography. The Refractive Index Detector functions with solvent with refractive indices between 1.00 and 1.75. The 2414 Refractive Index Detector can measure extremely small changes in refractive index as small as 7 x 10-9 by detecting the difference in the amount of light falling upon each of the elements of the dual-element photodiode.In refractive index, detection peaks can be either positive or negative, therefore it is essential that the polarity of the detector signals can be configured and changed when acquiring data. The polarity parameter is used to invert the sign of the refractive index data.

Photodiode Array (PDA) Detector computes absorbance by subtracting the dark current on reference spectrum from the acquired spectrum. Photodiodes lose charge over time even when they are not exposed to light. The amount of charge lost is called dark current. At the start of a chromatographic run, the 2996 Detector closes the shutter to take a dark current reading for each diode. The shutter closes after the exposure time is calculated and stays closed for the same interval as the exposure time. The detector subtract the dark current values from the current values recorded during absorbance measurements for both the sample and the reference spectra.

The 2996 Detector calculates the absorbance for each diode at the end of each exposure time. Absorbance is based on the principles of Beer’s Law:

 A = εlc

Where: A = absorbance, ε = molar absorptivity, l = path length (1.0 cm in the 2996 Detector normal flow cell), c = molar concentration.

Beer’s Law applies only to well-equilibrated dilute solutions. It assumes that the refractive index of the sample remains constant, that the light is monochromatic, and that no stray light reaches the detector element. As concentration increases, the chemical and instrumental requirements of Beer’s law may be violated, resulting in deviation from (absorbance versus concentration) linearity. The absorbance of mobile phase can reduce the linear range.

The molecular weight of a polymer is a prime importance in its synthesis and application. The interesting and useful mechanical properties that are uniquely associated with polymeric materials are a consequence of their high molecular weight. Polymers differ from the small-sized compounds in that they are polydisperse or heterogeneous in molecular weight. Even if a polymer is synthesized free from contaminants and impurities, it is still not pure substance in the usually accepted sense. Polymers are mixtures of molecules of different molecular weight. The reason for polydispersity (PDI) of polymers lies in the statistical variations present in the polymerization processes. The following average molecular weights are determined:

1. The number-average molecular weight M_n.

M_n = ∑N_xM_x

Where N_x is the mole-fraction (or the number-fraction) of molecules whose weight is M_x.

2. The weight-average molecular weight M_w.

M_w = ∑w_xM_x

Where w_x is the weight-fraction of molecules whose weight is M_x.

In addition to the different average molecular weights of a polymer sample, it is frequently desirable and necessary to know the exact polydispersity (PDI).

                  PDI = M_w/M_n

Gel permeation chromatography (GPC) involves the permeation of a polymer solution through a column packed with microporous beads of crosslinked polystyrene. The packing contains beads of different-sized pore diameters. Molecules pass through the column by a combination of transport into and through the beads and through the interstitial volume (the volume between beads). Molecules that penetrate the beads are slowed down more in moving through the column than molecules that do not penetrate the beads; in other words, transport through the interstitial volume is faster than through the pores. The smaller-sized polymer molecules penetrate all the beads in the column since their molecular size (their hydrodynamic volume) is smaller than the pore than the pore size of the beads with the smallest-sized pores. A larger-sized polymer molecule does not penetrate all the beads since its molecular size is larger than the pore size of some of the beads. The time for passage of polymer molecules through the column decreases with increasing molecular weight.

Please contact Krystyna Brzezinska (kbrzez@mrl.ucsb.edu) to schedule training. Before training starts please read MANUAL.