While you are looking for 0 when calibrating, by using ro or ro/di you will get less accurate results when you actually measure your tank water and are looking for 35ppt for example. When calibrated with ro or ro/di vs the 53ms pinpoint calibration solution you will be .015 off where you really are. That amount is not too bad but when doing hypo it can actually be the difference between an effective salinity and a non-effective salinty.
Here is a more thorough explanation;
Tips on Calibrating a Refractometer
Despite the fact that many refractometers sold to aquarists recommend calibration in pure water, such a calibration alone will not ensure accuracy for the reasons described above. So my recommendation for calibration is as follows:
1. First calibrate the refractometer in pure freshwater. This can be distilled water, RO (reverse osmosis) water, RO/DI water, bottled water and even tap water with reasonably low TDS (total dissolved solids). Calibrating with tap water that has a TDS value of 350 ppm introduces only about a 1% error in salinity, causing readings in seawater to read a bit low. So 35 ppt seawater (specific gravity = 1.0264) will read to be about 34.7 ppt, and will show a specific gravity of about 1.0261.
This calibration should ordinarily be carried out at room temperature using an ATC refractometer. The directions with some ATC refractometers insist that the calibration be carried out at a specific temperature, but I've never understood how that could matter and I would not worry about it. If the refractometer is not an ATC refractometer, then careful temperature control or correction is necessary, and such corrections are beyond the scope of this article.
Calibration is usually performed by putting the freshwater on the refractometer, letting it sit for at least 30 seconds so it comes to the same temperature as the refractometer, and adjusting the calibration screw until it reads a value appropriate for freshwater (e.g., refractive index = 1.3330, salinity = 0 ppt, specific gravity = 1.0000). Normally, this step is a quick and easy procedure, and may often be all that is required IF the refractometer has been verified to have passed the second calibration step below at least once. This is an offset calibration, as described above.
2. The second step in calibration should be performed at least once before relying on a refractometer to accurately measure the salinity of a reef aquarium. This step involves testing it in a solution matching the refractive index of 35 ppt seawater (or some similar solution near the range of measurement). Remember to let it sit for at least 30 seconds so it comes to the same temperature as the refractometer. Suitable commercial and do-it-yourself standards were described earlier in this article. Using one of them, place a drop onto the refractometer and read the value. If it reads approximately 35 ppt, or a specific gravity of 1.0264, or a refractive index of 1.33940, then the refractometer is properly calibrated and is set to go.
If it does not read correctly, and is off by an amount that is significant relative to your salinity precision requirements, then you need to recalibrate it using this second fluid. I suggest that a salinity error of ± 1 ppt or a specific gravity error of ± 0.0075 is allowable. If the refractometer is off significantly, and you used a do-it-yourself standard made with crude techniques such as Coke bottles, a good next step might be to buy a commercial standard.
To correct errors using these seawater standards, simply adjust the calibration screw on the refractometer until it reads the correct value for the standard (35 ppt, or a specific gravity of 1.0264, or a refractive index of 1.33940). This type of slope calibration makes the refractometer suitable to read solutions whose salinity is close to seawater's. After such a calibration, refractometers may not read freshwater correctly.
Again, despite the claims in the directions of some refractometers to have the standard at a particular temperature, when calibrating an ATC refractometer with this seawater standard, I'd just use it at room temperature.
If you are using a refractometer for hyposalinity, such as when treating a sick fish, I'd just use one calibrated in freshwater, because that is closer in salinity than seawater to the hyposaline solution usually used (say, specific gravity = 1.009). A new standard for hyposalinity can also be made by mixing one part 35 ppt seawater and two parts freshwater, but that is probably overkill.
