Detector D.R. = f (m A ); Sample D.R. = f (c A M.Ph . ) Specific c A Injection System V inj M.Ph. D.R. V inj for a given C A m A C A D.R. Sensitivity thus V inj Sensitivity
Usual sample preparation procedures Solid phase extraction Liquid-liquid extraction (SPE) (LLE) Aqueous non- Aqueous non- Aqueous miscible miscible phase miscible phase organic phase Direct Dilution Direct Direct small with water small large volume volume volume injection injection injection (SVI) (SVI) (LVI) Large volume Solvent injection evaporation (LVI) Residue re- dissolution SVI / LVI Reversed Phase Liquid Chromatography (RPLC)
For achieving Large Volume Injection (LVI) in liquid chromatography the sample diluent should be entirely miscible to and weaker than the mobile phase composition at the beginning of the separation process.
Column: Zorbax Eclipse C-8 150 mm x 4.6 mm x 5 m; 100 OH mAU Mobile Phase: ACN : aq. 0.1% H 3 PO 4 = 4/6 (v/v); C(CH 3 ) 3 Flow Rate: 1 mL/min; Detection: UV – 291 nm; Diluent (D) = i -octane; V inj = 5 – 600 L 80 12 O 11 CH 3 10 9 8 60 7 k (BHA) 6 5 4 40 3 600 L 2 500 L 1 400 L 0 20 0 100 200 300 400 500 600 700 300 L V inj ( L) 200 L 100 L V. David, C. Barcutean, C. Georgita, A. Medvedovici, 5 L Non-miscible solvent LVI-HPLC/DAD method for min 0 determination of butylated hydroxyanisole in lovastatin 0 10 20 and simvastatin pharmaceutical formulations, Rev. Roum. Chim. , 5, 445-451 (2006).
k A (Retention Factor) = K A (Partition Constant) x V S.Ph. / V M.Ph. D is practically totally partitioned in the S.Ph. and exhibits similar properties; consequently: real = V S.Ph. + V inj V S.Ph. Diluent k A = K A x (V S.Ph. + V inj Diluent ) / V M.Ph. Diluent than k A V inj
A (D) A (M.Ph.) [1] A (M.Ph.) + L (S.Ph.) A*L (S.Ph.) [2] if assuming [D] >> [A] and log P D > log P A i D (M.Ph.) + L (S.Ph.) D i *L (S.Ph.) [3] [1] ; [2] ; [3] ; k A = K A x V’ S.Ph. /V M.Ph. the V’ S.Ph. available for A is a fraction of V S.Ph. , D , where is a constant more precisely (V S.Ph. - V), where V = x V inj D k A = K A x V S.Ph. /V M.Ph. – (K A x /V M.Ph. ) x V inj A. Medvedovici, Victor David, Vasile David, C. Georgita, Retention phenomena induced by LVI of solvents non-miscible with the mobile phase in RPLC, J. Liq. Chromatogr. Relat. Technol. , 30, 199-213 (2007).
S.Ph. A C Diluent S 0 S 1 M.Ph. u S 2 S Analyte B D u AT th x (1-V inj D /V 0 x S 0 /(S 0 -S 1 )) k A = k A
Rule of five for „on-line RSLE” 1D exhibits an increased chromatographic retention compared to front > k A ); target compounds (k D 2Solubility of D in the M.Ph. should be as low as possible; 3The initial chromatographic resolution supports the “apparent” reduction of the column length (affecting selectivity). 4D plug from a previous injection is already eliminated from the column before starting a new separation process; 5Fingering effects due to different viscosities (D vs. M.Ph.) need attention and should be controlled;
Solute: Metoprolol (log P = 1.88) Diluents: Butyl acetate (log P = 1.78) Carbon tetrachloride (log P = 2.83) 1-Octanol (log P = 3.00) H N Cyclohexane (log P = 3.44) n-Hexane (log P = 3.9) OH Injection Volumes: 1, 5, 10, 20, 50, 75, 100 �L O Chromatographic Columns: Zorbax Eclipse XDB C-8 (150 mm x 4.6 mm x 5 m); Zorbax Eclipse XDB C-18 (150 mm x 4.6 mm x 5 m); Zorbax C-18 Stable Bond AQ (150 mm x 4.6 mm x 5 m); Chromolith Performance C-18 (100 mm x 4.6 mm); Betasyl Phenyl (150 mm x 4.6 mm x 5 m); Luna PFP (100 mm x 4.6 mm x 3 m); Mobile Phase: O Isocratic Elution Organic Solvent: ACN Aqueous Solvent: 50 mM HCOONa + 0.2% TEA at pH = 3.5 with HCOOH Composition: Organic / Aqueous Solvents = 10/90 (v/v)
C8 100 L 1 mAU M. Ph. BuOAc CCl 4 Oct cHex Hex 1 min
21.00 M.Ph. C8 column BuOAc CCl4 20.00 Oct cHex 19.00 Hex Retention factor (k) 18.00 y = -0.0437x + 19.832 R 2 = 0.999 17.00 y = -0.0294x + 19.831 R 2 = 0.9988 y = -0.0328x + 19.945 16.00 R 2 = 0.9991 y = -0.0327x + 19.814 R 2 = 0.9939 15.00 y = -0.0322x + 19.835 R 2 = 0.9982 14.00 0 20 40 60 80 100 120 Injected volume (uL)
0.0000 -0.0100 Slope of k=f(Vinj) relationship Panta medie k=f(Vinj) -0.0200 -0.0300 -0.0400 XDB C18 XDB C8 -0.0500 SB AQ C18 Chromolith C18 Betasil Phenyl PFP -0.0600 1.5 2 2.5 3 3.5 4 4.5 Log P diluent log P Diluent
Slopes of the regression k = f(V inj ) Column/ Diluent Flow SB AQ Chromolith Betasil XDB C18 XDB C8 PFP rate C18 C18 Phenyl (mL/min) 0.5 - - - - - -0.0267 0.75 - - - - - -0.0323 1 -0.0408 -0.047 -0.0181 -0.0342 -0.0133 -0.0304 1.25 - - - - - -0.0317 1-Octanol 1.5 -0.0364 -0.0465 -0.0173 -0.0358 -0.0132 -0.0315 2 -0.0374 -0.0452 -0.0183 -0.0319 -0.0125 - 2.5 -0.0397 -0.0402 -0.0161 -0.0328 -0.0127 - 3 -0.0412 -0.0398 -0.0177 -0.0338 -0.012 - Mean -0.0391 -0.0437 -0.0175 -0.0337 -0.0127 -0.0305 St. Dev. 0.0021 0.0035 0.0009 0.0015 0.0005 0.0022 RSD% 5.4 8.0 5.0 4.4 4.2 7.3 0.5 - - - - - -0.0316 0.75 - - - - - -0.0345 1 -0.0370 -0.0306 -0.0183 -0.0178 -0.0147 -0.0342 1.25 - - - - - -0.0326 Cyclohexane 1.5 -0.0406 -0.0345 -0.0189 -0.0169 -0.0167 -0.0341 2 -0.0400 -0.0327 -0.0188 -0.0158 -0.0181 - 2.5 -0.0426 -0.0315 -0.0196 -0.0156 -0.0182 - 3 -0.0430 -0.0336 -0.0198 -0.0160 -0.0201 - Mean -0.0406 -0.0326 -0.0191 -0.0164 -0.0176 -0.0334 St. Dev. 0.0024 0.0016 0.0006 0.0009 0.0020 0.0012 RSD% 5.9 4.8 3.2 5.6 11.4 3.7
0 BuOAc CCl4 1-Octanol -0.01 Cyclohexane Hexane -0.02 Slope -0.03 -0.04 -0.05 C18 AQ Phenyl PFP C18 C8 -0.06 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 H
y = -7E-06x 2 + 0.0012x - 0.0665 0 R 2 = 0.9996 0 10 20 30 40 50 60 70 y = -2E-06x 2 + 0.0003x - 0.0257 R 2 = 0.9941 -0.01 y = 0.0003x - 0.0243 R 2 = 0.9726 y = -3E-06x 2 + 0.0006x - 0.042 Slope of k = f(Vinj) -0.02 Panta k = f(Vol.inj.) R 2 = 0.9823 y = 0.0002x - 0.0173 R 2 = 0.9922 -0.03 y = 0.0004x - 0.0455 R 2 = 0.9863 -0.04 XDB C18 SB AQ C18 -0.05 Chromolith C18 XDB C8 Betasyl -0.06 Temperature ( o C) PFP Temperatura
2.800 y = 1420.6x - 2.1356 Van't Hoff plots R 2 = 0.9926 2.700 Diluent: 1-Octanol Column: SB AQ C18 y = 1410x - 2.104 2.600 R 2 = 0.9941 2.500 y = 1413.5x - 2.1235 R 2 = 0.993 2.400 ln (k) y = 1426.1x - 2.1743 2.300 R 2 = 0.9924 y = 1424.3x - 2.2083 2.200 1 uL R 2 = 0.9931 5 ul 2.100 y = 1436.7x - 2.2855 10 ul R 2 = 0.9935 20 ul 2.000 50 ul y = 1435.6x - 2.3159 1.900 75 ul R 2 = 0.9929 100 ul 1.800 0.0029 0.003 0.0031 0.0032 0.0033 0.0034 0.0035 1/T (1/K)
8000 SB AQ C18 7000 6000 N (Foley-Dorsey) 5000 M.Ph. BuOAc CCl4 4000 Oct cHex 3000 Hex 2000 1000 0 0 20 40 60 80 100 120 Injection Volume (uL)
3.500 SB AQ C18 3.000 Peak Assymetry (10%) 2.500 M.Ph. BuOAc CCl4 2.000 Oct cHex Hex 1.500 1.000 0.500 0 20 40 60 80 100 120 Injection Volume (uL)
1. The on-line RP-SLE model fits better to experimental observations compared to the adsorption model. 2. The non-miscibility of the diluent with the mobile phase seems to play the most important role compared to the relationship between the hydrophobic characteristics of the diluent and analytes. 3. The kinetic of the LLE process is less important for analytes having an increased hydrophobic character, as long as the “free” stationary phase will refocus them. 4. The kinetic of the LLE process becomes important for analytes having hydrophilic character, as long as the “free” stationary phase will not refocus them. 5. The process will be better controlled by means of a gradient elution scenario: rich aqueous composition fix the diluent front, next the increase of the organic modifier produces LLE and control refocusing.
SO 3 Na O O O + N Br N N O N N O O N N H + NH 2 N Cl Metamizole sodium Metamizole Imp. C Fenpiverine Bromide Pitofenone Hydrochloride (MTZ) (MTC) (PTF) (FPB) log Dow (pH=3) Log Dow (pH=3) Log Dow (pH=3) Log Dow (pH=3) -2.24 0.76 -0.56 0.66 500 mg/mL (max. 3.5% from MTZ) 20 ug/mL 2 mg/mL 500 X dilution (17.5 mg/mL) 25 X dilution IP-LLE+RP-SLE 25 X dilution Polar Compounds! Opposite ion pairing characteristics! Tailing favored by increased interaction to residual silanols! Quantitatively uncompensated mixture: (MTZ/FPB = 1/25,000; PTF/FPB = 1/100; MTZ/PTF = 1/250) T. Galaon, M. Radulescu, V. David, A. Medvedovici, use of an immiscible diluent in ionic-liquid / ion-pair LC for the assay of an injectable analgesic, Cent.Eur. J. Chem. , 10(4), 1360-1368 (2012).
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