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 of three

5
Imperfects of parametric deconvolution Peak
broadening and column 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).