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May/June 2013


• using at least three different lots of column media and solvents


• checking to see what effect the dwell volume can have on the assay by running on different manufacturer’s pumps.


The use of predictive software tools can substantially help here to reduce valuable laboratory time; however there is an initial cost implication to going down this route.


References


1. S. Scott “Chemical Chaos” Oxford Science Publications (1991)


2. E. Lorenz, J. Atmospheric Sci. 20 (2) 130–141 (1963)


3. J. Colloid and Interface Sci., 26, (1) 62-69 (1968) W. Stöber, A. Fink, E. Bohn


Figure 4: The new assay developed at low pH has a much greater stability. Experimental conditions:


Mobile phase:A – 30 mM KH2PO4 (pH= 2), B – MeCN, Gradient : 10 to 80% B in 10 min, Flow rate: 0.2mL/min, Temperature: 30 °C, UV Detection: 210nm, Injection volume: 5µL Analytes:


1. Doxylamine (B), 2. Hydroxyisophthalic acid (A), 3. Benzamide (N), 4. Doxepin (B) 5. Flavone (N), 6. Fenoprofen (A)


method was developed at a lower pH, Figure 4, and tested on three different columns from three different batches. The resulting chromatography needed further optimisation to separate all of the components but what the chromatogram shown which only differs from the original method in the pH that was used, does show is that the chromatography is stable from one column to another, and in particular the basic compounds which were causing the issue with the original method.


The use of predictive software [7, 9-12] can make method optimisation and assay stability determination a much less onerous task, virtually eliminating the need for lots of experimental data to ensure that the assay is stable. There are many commercially


available products on the market, with most based on well understood theoretical models and for the majority of experimental arrangements these work incredibly well.


Conclusion


To avoid assay instability it is important that the chromatographic separation is stable to small changes that may not be within the control of the analytical scientist. Thus it is important to check the stability of the assay by varying a range of parameters for the final assay conditions. This should include:


• varying the temperature by ±5°C • varying the pH by ±2 units


Fast Separations of Inorganic Ions in Water Samples


Thermo Fisher Scientific is pleased to announce a new technical note about the determination of inorganic anions in water samples using our 4 µm ion-exchange column (Thermo Scientific™ Dionex™ IonPac™ AS18-4µm column). Technical Note 127: Efficient and Fast Separations of Inorganic Anions in Water Samples Using a 4 µm Particle Size Microbore Column with a High-Pressure Ion Chromatography System presents the advantages of using the small-particle size microbore (2 mm i.d.) Dionex IonPac AS18-4µm column, in combination with the high-pressure capillary Ion Chromatography system, Thermo Scientific™ Dionex™ ICS-5000+HPIC™ system to obtain fast and efficient separation of inorganic anions in municipal drinking and wastewater samples.


In 1993, the U.S. EPA Method 300.0 (A) defined the use of the Thermo Scientific™ Dionex™ IonPac™ AS4A anion-exchange column to determine inorganic anions in environmental waters using a manually prepared carbonate/bicarbonate eluent and suppressed conductivity detection. With the introduction of Reagent-Free™ ion chromatography (RFIC™) systems, hydroxide-based eluents were electrolytically generated inline on anion-exchange columns optimized for hydroxide chemistry. The Dionex IonPac AS18 column was developed as an alternative to the AS4A column; the AS18 is designed for use with EPA Method 300.0 (A) using Electrolytically Generated hydroxide eluents. These smaller resin particles allow an optimal combination of fast speed and high resolution.


This technical note and many others can be found at www.thermoscientific.com/dionex under the Documents tab.


4. Porous silica, its properties and use as support in column liquid chromatography, K. K. Unger, Elsevier Scientific Pub. Co., 1979


5. US Patent 3943072, A. R. Thomson and B. J. Miles, United Kingdom Atomic Energy Authority, London (EN)


6. J. Chrom. A, D.V. McCalley, 1217 (6) 858- 880 (2010)


7. www.chemicalize.org


8. Advanced Drug Delivery Reviews. 23, (1–3) 1997, 3–25, C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney


9. Cs. Horváth, W. Melander, I. Molnár, J. of Chromatogr. 125, 129-156 (1976)


10. S. Fekete, J. Fekete, I. Molnár, K.Ganzler, J. Chrom. A 1216, 7816-7823 (2009).


11. I. Molnár, H.-J. Rieger, K.E. Monks, J. Chromatogr. A 1217, 3193-3200 (2010).


12. A. Vailaya, Cs. Horváth, J. Chrom A. 829 1 (1998)


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