Heterogeneity in Molecular Dimensions (V e ): mass molar fraction profiles
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1 molar fraction eterogeneity in Molecular Dimensions (V e ): mass molar fraction profiles R = e, i 3 V 4 π e, i 1 3 m _ R e D _ d n _ R e D _ d sphere equivalent radii distribution (R e D) of SEC-separated glucans mass fraction mass(i) vs V ret m_red_d differential mass fraction distribution mol(i) vs V ret n_red_d differential molar fraction profile Re [nm]
2 Mobility of Molecular/Supermolecular Structures translational Diffusion coefficient D T D T converted to dimension of coherent segment lengths mass fraction LS-intensity fraction coherence length [nm] D mass modulated fractions molar mass of glucans T kb T = 6πη R coherence length l coh [nm] V 2 - (intensity) modulated fractions apparent molar masses of supermolecular structures
3 Mobility of Molecular/Supermolecular Structures a1t8 a1t7 a1t6 a1t5 a1t4 a1t3 a1t coherence lenghts [nm] homogeneous scb glucan cerial starch amaranth up to C in DMS a1t w1t8 w1t7 w1t6 w1t5 w1t4 w1t3 w1t radius [nm] scb + lcb heterogeneous glucan cereal starch wheat up to C in DMS w1t1
4 Preparative SEC pooling (1) fragmentation fractionation (2) V(excl) fraction 1 fraction 2 V(tot) V(excl) V(tot) ev [1/mL] ev [1/mL] Vret [ml] starch granules / potatoe species stara Vret [ml] debranched fraction 1 debranched fraction 2 Sephacryl S-200 / S400 / S-500 / S-1000 ( x 1.6 cm) eluent: M Na flowrate: 0.67 (1), 0.40 (2) ml/min V excl =220 ml - V tot =510 ml
5 Semi-preparative SEC: Branching Analysis ev [1/mL] V_ret [ml] E_640 / E_525 non-destructive approach SEC separtion of heterogeneous lcb+scb glucans iodine staining / complexation of fractions spectroscopy (E 640 lcb / E 525 scb) destructive approach fractionation due to different solubilities controlled step-by-step fragmentation fragment analysis (PLC, SEC) 60 % n-bu precipitate lcb % dpn Glc # seg helical full turns br% molecule C - chains x 2-15 A + B 55 A - chains x 2 B - chains x 7 40 % Me precipitate scb % dpn Glc # seg helical full turns br% x x x 7
6 Population Analysis from SEC-elution profiles 0.15 heterogeneous scb / lcb glucan (cerial starch: millet) (normalized) SEC-elution profile of e.g. mass fractions ev [1/mL] y = i find a minimum of base function = populations σ A 2π exp (M - M Mp ) 2 2σ LSQ-linear combination of base-functions to match the SEC-elution profile V_ret [ml] i = Min (7) 7 i= 1 y SEC = Min i profile
7 Analytical SEC + multiple inline detection V_ret [ml] excluded volume fractionation V e (i) from SEC details on excluded volume fractions V e (i) from triple-detection of mass, scattering, viscosity detailed excluded volume Distribution (m_v e D, n_v e D) from SEC-triple-detection - profiles mass by refractive index DRI ( ) scattering intensity as Rayleigh Θ 0 ( o ), specific viscosity from differential pressure (dp) / inlet pressure (ip) ratio ( ).
8 Molecule Conformation from SEC-mass, visc, LS lg(eta_int) lg(m) lg(ve) lcb-glucan potato a = 0.27 V_ret [ml] lg([eta]) [ml/g] [ η ] = K. V e M = ip. md a mc lg(eta_int) lg(m) lg(ve) potato waxy maize V_ret [ml] scb-glucan waxy maize a = 0.29 K = 0.22 [ml mol g -2 ] 0.4 K = 0.16 [ml mol g -2 ] lg(m) [g/mol]
9 SEC mass / LS: apparent molar masses superimposed molecular + supermolecular structures V e = ip. md mc 8.50 period of thermal stress dissolution process in 90 C [h] scb-starch glucan M app [g/mol] 8.00 lg(m) [g/m] V_ret [ml]
10 Chemistry + SEC-dual-detection absolute and de facto molar masses by combining SEC elution profiles of mass fractions [g/l] + molar fractions [mol/l]
11 100 µm 100 µm Starch Glucans reductive pyridyl-amination (PA) of starch free terminal hemiacetals: AP-glucans R + 2 N N - 2 R C N N + 2 R C N N diss. of g glucan + 500mg 2-aminopyridine + 1mL 2 for 3 60 C neutralization to p 6-7 with 4 M Cl for 1 60 C reduction to stabilized secondary amine with cyanoboro hydride for 2 60 C precipitation of AP-Glc n in Me excess acetone washing N 2 -steam drying
12 AP-Glucans: absolute molar concentrations extinction E 380 nm, excited at 315 nm wavelength [nm] PA-maltose [nmol/ml] UV/vis spectra of aminopyridyl (AP)-maltose ( ) and aminopyridyl (AP)-dextran ( ) fluorimeter 380 nm with AP-maltose molecular weight ( C N 2 ) 258 g/mol
13 AP-Glucans: SEC-mass/molar Analysis mass fractions [g/l] SEC elution profile molar fractions [mol/l] SEC elution profile
14 SEC-mass/molar Analysis of AP-Glucans absolute de facto SEC molecular weight calibration
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