2. The major issue with this is for you to ensure that your system can handle the pressures that will be generated with the column and method you plan to use. You will need to know the dimensions of the column (internal diameter - aka ID and length), the particle size and the flow rate you plan to use. Also, you need to consider the binding capacity of the column. Ensure that if your sample is large that your column can bind the entire sample (need a larger column) or if your sample is small that your sample resolution is maintained (need a smaller column).
Another thing to consider when you are choosing your column is to make sure there isn’t a large disparity between the size of your flow cell and the ID of your column. If the difference between the two is great it can cause dispersion which can lead to unexpected peak alterations which can impact the LC-MS results.
In the laboratory that I work at we have our choice of different LC-MS systems... but If I could make a recommendation based on how we handle our iTRAQ samples, it would be to use an offline SCX-MUDPit protocol for cleaning your sample prior to LC-MS. The advantages of this are:
- simpler to use and much less room for error, this is extremely important especially if you do not have much HPLC experience
- Tighter elution windows for your fractionation, your samples will typically be in smaller volumes than if you ran them on an HPLC
- Cost. A box of SCX SpinTips cost a lot less than an HPLC column. Furthermore, if you are running this on the HPLC you will lose a lot of sample while you are developing your method, this could require you to repeat your iTRAQ several times. Just setting up proper fraction collection windows can lead to a lot of trial and error.
3. If you don’t have a fraction collector you can certainly do it by hand... however, you want to make sure that your fractions are collected accurately at highly specific intervals. As far as collection vessels are concerned you just need to make sure that they can hold the volume of your fraction (flow rate x time of elution window).