Agilent poroshell 300sb c18 column

The casein fractions (κ-CN, α S1 -CN, α S2 -CN, β-CN, α-LA, β-LG A, β-LG B, and total casein) were identified and quantified (in duplicate) in the inoculated milk samples by HPLC. Milk samples (200 µL) were diluted in 3,780 µL of dissociating buffer (7 M urea and 20 mM Bis-Tris propane, pH 7.5). Twenty microliters of mercaptoethanol were added to the diluted samples, after which they were filtered through 0.22-µm filters. The equipment used was an Agilent 1200s system (Agilent Technologies, Santa Clara, CA) with a quaternary pump and a multiwavelength detector. The casein fractions were separated in the reversed-phase mode using an Agilent Poroshell 300SB C18 column (2.1 mm × 7.5 mm; Agilent Technologies). The gradient elution and peak detection were performed according to the method of Mounsey and O'Kennedy (2009) (link).

Duplicate bulk tank milk samples and duplicate milk line samples were analyzed daily for fat, protein and lactose composition and SCC using a Milkoscan 203 (Foss Electric, Hillerød, Denmark) .
Duplicate bulk storage tank milk samples were submitted for wet chemistry analysis of nitrogen fractions after storage for 0, 48 and 96 h. The percentage total protein, NPN, and noncasein nitrogen content of the milk samples were determined using the Kjeldahl method [methods 20-3 (IDF, 2004b ), 20-4 (IDF, 2001 ), and 29-1 (IDF, 2004a) , respectively] using a Tecator Digestor Auto and Kjeltec 8400 distiller (Foss Electric).
The protein composition of duplicate bulk storage tank milk samples was quantified daily (0, 24, 48, 72, and 96 h) by HPLC using the method described by Mounsey and O'Kennedy (2009) . Briefly, 200 μL of milk was diluted in 3,800 μL of dissociating buffer (7 M urea and 20 mM Bis-Tris propane, pH 7.5), to which 5 μL of 2-mercaptoethanol was added, before filtering through a 0.22-μm filter. Separation of the milk protein fractions was achieved in reverse-phase mode, using an Agilent Poroshell 300SB C18 column (2.1 × 75 mm; Agilent Technologies, Santa Clara, CA). The HPLC equipment consisted of an Agilent 1200s with quaternary pump and multi-wavelength detector. Gradient elution and peak detection were performed according to the method of Mounsey and O'Kennedy (2009) .