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Lot to lot variations on BioSep4000 (2)

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Application Detail (App ID: 18897)

Column: BioSep™ 5 µm SEC-s4000 500 Å, LC Column 300 x 7.8 mm, Ea

Part No:00H-2147-K0

Dimensions:300 x 7.8 mm ID

Elution Type:Isocratic

Elution A:100mM Phosphate buffer pH 6.8

Gradient Profile:

Step No.	Time (min)	Pct A
1	0	100

Flow Rate:1 mL/min

Col. Temp: ambient

Detection: UV-Vis Abs.-Variable Wave.(UV) @ 220 nm (22°C)

Note:Application Focus: Investigations on lot to lot variations for BioSep 4000

The purpose of this application is to show lot to lot reproducibility (or variation) between BioSep 4000 media lots. GFC columns (300 x 7.8 mm) from three different silica batches were overlaid to show retention and recovery differences. A GFC standard focused across the BioSep 4000 optimal resolution window was used (using commercially available proteins) and standard running conditions were utilized to best represent "typical" running conditions (100 mM phosphate, pH 6.8, 1mL/min.).

When batch specific chromatograms are overlaid, one can see that the standard chromatograms look very similar batch to batch. Both peak retention times and protein recoveries are very similar indicating that the BioSep 4000 is a very reproducible media batch to batch. Of specific note, batch to batch recoveries of every protein looks equivalent except for thyroglobin which displays variability across repetitive injections possibly due to solubility issues, putting light to the challenge in finding standard proteins for larger molecular weight ranges. Other observations of note include the noticeable resolution between Ig-M and the aggregate peak, this resolution demonstrates the utility of the BioSep 4000 column in resolving peaks as high as one million daltons molecular weight. A final note relates to the uridine void marker peak. In some cases a low molecular weight contaminant from Ig-M is partially resolved; in other chromatograms this impurity coelutes with uridine making it appear as a fronting peak. This further illustrates the challenges in finding acceptable standard proteins for large pore sized GFC columns. In the end, the BioSep 4000 demonstrates excellent performance as a GFC column, especially for high molecular weight proteins which have their specific challenges.

Analyte Details

Analyte Detail not available

High MW impurity

IgM

Thyroglobulin

IgA

beta-Amylase

BSA

Ribonuclease A

Uridine