Within reason, you can basically use any buffer you like with Sephacryl S-100, provided that is suits your purpose in terms of protein/enzyme stability, and phosphate-buffered saline sounds ideal.
There are sometimes a small amount of positively or negatively charged groups associated with a gel-filtration resin, introduced during the manufacturing process, and it is recommended to have some salt present (100mM KCl used to be recommended) in order to counteract anion-exchange and/or cation exchange effects. In addition, at very high salt concentrations (1M ammonium sulphate or higher), proteins may bind to the resin by a hydrophobic interaction. Furthermore, one or more of your target molecules may be unstable at extremes of pH. NADH, for example, is unstable in acid.
In other words, you want to set up the experiment where the target molecules are stable, and where separation on the column is by 'molecular sieving' only.
A very common method of measuring protein concentration is to measure the A-280 of each fraction (the absorbance at 280nm). The has the big advantage of being reasonbly sensitive AND is non-destructive (you can fully recover the sample after the measurement). All you need is access to a spectrophotometer and a quartz cuvette!
In addition, with (pure) BSA the method may be made quantitative as the A(1%, 280),sometimes called the E(1%,280), is known. From memory I think this is about 0.68 for BSA. That is, a 1mg/ml solution of (pure) BSA will have an absorbance at 280nm of 0.68. (The 'rule of thumb' for impure protein preparations is that a 1mg/ml solution has an absorbance of 1. But note that many compounds absorb at 280nm, including DNA)
You can, if you wish, calculate the A(1%, 280) from the amino acid sequence of the protein of interest. The refs (Perkins; Gill & von Hippel) are given below.
It should be possible to detect B12 by a similar method, perhaps even at 280nm, but an absorbance spectrum may yield a more sensitive/specific wavelength.
You don't give the pH of the PBS (!important), or the dimensions of the Sephacryl column.
Long, narrow columns work best, but you probably already know that.
Gel Filtration by Reiland (Methods in Enzymology, 1971). A great article
Protein Purification: Principles and Practice by RK Scopes (Springer Advanced Texts in Chemistry) 3rd Edition
Sober, E. K. & Sober, H. A. (1970). Molar extinction coefficients and E (1%, 280) values for proteins at selected wavelengths of the ultraviolet and visible region. In Handbook of Biochemistry. Selected Data for Molecular Biology, 2nd edn. Sober, H. A., Ed. pp C-71 - C-98. The Chemical Rubber Company, Cleveland, Ohio.
Perkins, S. J. (1986). Protein volumes and hydration effects. The calculation of partial specific volumes, neutron scattering matchpoints and 280-nm absorption coefficients for proteins and glycoproteins from amino acid sequences. Eur. J. Biochem. 157, 169 - 180. [Pubmed] [pdf]
Gill, S. C. & von Hippel, P. H. (1989). Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182, 319 - 326. [published erratum appears in Anal. Biochem. (1990) 189, 283].