Superresolution Chromatography

1. Superresolution Chromatography E.L.Kosarev - P.L.Kapitza Institute for Physical Problems, RAS http://www.kapitza.ras.ru/people/kosarev/home.htm K.O.Muranov - N.M.Emanuel Institute of Biochemical Physics, RAS http://kmuranov.euro.ru

2. Chromatographic analysis Chromatography: • Adsorption/Partition • Affinity • Ion-exchange • Size-exclusion

3. Peaks’ overlapping problem • Peaks overlapped form the joint peak

4. Imperfects of parametric deconvolution • Supposition - Peak shape can be described analytically (Gaussian, Lorenz, etc.) • Parametric deconvolution needs: • peak position • peak wide • peak amplitude. • Unknown peak characteristic causes an error. For instance, two peaks were identified instead ofthree

5. Imperfects of parametric deconvolutionPeakbroadeningandcolumn voiding The main factors • Diffusion • Non-specific interaction • System overdampening (irreversible adsorption, filter contamination, plunger damage, etc.)

6. Problems: • the shape of a real chromatography peak could not be approximated exactly with any function • the parametric technique cannot give reliable results

7. We suppose: the problem of overlapping peaks’ separation could be accurately solved with the use of a nonparametric method

8. Nonparametric method • The peak's shape is determined directly from the separation of an individual compound and can be called the point spread function of a chromatographic column • If the shapes of these peaks are the same throughout the entire working range of the device, then chromatogram is a distribution convolution with the peak of this shape • the point spread function includes all factors influenced the separation

9. Nonparametric method • The decomposing a complex spectrum into the same components is achieved by solving an integral convolution equation • For this equation: • the input data is the chromatogram • the convolution operator kernel is the point spread function of the chromatograph column

10. Analysis of chromatography separation data with the RECOVERY software package Method • A protein mixture of known composition (bovine serum albumin monomer, dimer, and trimer) was separated by gel filtration for obtaining of heavily overlapping peaks. • The data was processed with the use of the RECOVERY software package • The result was compared with the finer separation data obtained with the use of the HPLC.

11. BSA chromatography and point spread function determination A - Elution profile of bovine serum albumin (BSA). • The blue arrow indicates unresolved peak • Red dashed lines mark the time interval when the BSA monomer fraction was collected B -Elution profile of the collected fraction • Absence of unresolved peak • Strongly pronounced a peak asymmetry

12. Recovering of the chromatographic separation data • A - the source BSA chromatogram; • B - point-spread function; • C - recovering result of the chromatographic separation data with the RECOVERY software package.

13. The RECOVERY result vs. HPLC separation A - data recovered with nonparametric approach B - Elution profile of HPLC separation (TSK G2000 SW Spherogel, 10mm X 600 mm, flowrate - 0.5ml/min) • Both Recovery software package and HPLC found the BSA monomer, dimer and trimer in protein mixture • Recovery shows the more precision result

14. Peak width and whole column limit- under investigation • Separation of the rat eye lens crystallins (magenta) • 10 kD - 1.5 MD limit • Peak width of the standard proteins (blue)

15. Conclusion: • the proposed method fundamentally improves the quality of the chromatographic separation • RECOVERY software package for the gel filtration data significantly increased the resolution of this method and exceeded the quality of the separation obtained with the HPLC technique • new possibilities are achieved through reasonable processing of the measured data with no complication in the instrumentation • cost of the used instrument complex for gel filtration ($1,000) is roughly 15-20 times lower than that for the HPLC setup ($20, 000).