We have HPLC-UV (Shimadzu) in combination with 2465 Waters ECD in our Lab. I want to do some sugar analysis using HPLC-ECD. Since both from different company so I believe I need to set up two different methods. I can set up the LC method at Shimadzu's lab solutions software but for the ECD detector I need to set up the method manually. I am struggling to do this. At first I want to try simple sugar analysis to check how it works. Please share if you have any experience on it or any valuable suggestions how can I proceed on?
Hi Dulali,
I guess your Shimadzu LC system has capabilities to do data acquisition via an A/D converter (for instance a Shimadzu CBM20 has the option to install a PC55N ADC card). So in that case you can use the analog output signal of the Waters 2465 ECD and acquire it in the Lab Solution software. You need to make a dedicated cable (BNC-to-PC55N connector to connect the devices).
For sugar analysis Anion-Exchange chromatography in combination with Pulsed Amperometric Detection (PAD) on a Au Working electrode is the method of choice. The PAD settings can be easily programmed in the 2465 ECD via the keyboard/display (You can access the PAD menu via the main menu by pushing the F3 button). In the 2465 you are able to program a three-step potential waveform suitable for the detection of carbohydrates. Below you find some useful links to Carbohydrate application notes from our website (Antec Scientific).
https://antecscientific.com/download/18/carbohydrate-applications/8297/220_002_08-carbohydrates-in-food-products
In the link below you will find all relevant application notes which can give you directions to develop your specific application:
hhttps://antecscientific.com/solutions/applications/carbohydrates/
Regarding PAD settings for carbohydrates, typically a 4-step potential waveform is used (the so called waveform A). Unfortunately the 2465 can only handle three step waveforms. This is not really a big problem because you can derive a 3-step pulse (a compromise) which comes very close to that of the 4-step waveform:
E1, E2, E3: 0.1V, -2.0V, -0.1V; t1, t2, t,3: 400 ms, 20 ms, 80 ms; ts = 200 ms
We used this as an alternative and have seen that it worked pretty well. You could also take a more classical 3-step pulse. An example is shown below, but this waveform leads to more wear of the Au working electrode:
E1, E2, E3: 0.05V, 0.75V, -0.80V; t1, t2, t,3: 500 ms, 130 ms, 120 ms; ts = 60 ms
Regarding the chromatography, please read the information in the introduction of the application note in the first link carefully. There are quite some things which you need to take care of, regarding the preparation of the mobile phase. You need to avoid the introduction of CO2 in your alkaline (NaOH) mobile phases. Dissolved CO2 will convert into Carbonate ions which will lead into a loss of retention and not reproducible chromatographic separation.
Hope it helps. Regards, Hendrik
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