The simplest way to enhance on Sensitivity is to increase V inj ! - PowerPoint PPT Presentation

Detector D.R. = f (m A ); Sample D.R. = f (c A ) Specific c A Injection System V inj amount of A loaded to column   1)  D.R. amount of A loaded to column (as c A is specific to the sample)   2)  V inj V inj   D.R.   3)  Sensitivity The simplest way to enhance on Sensitivity is to increase V inj !

The Golden Rule of the Thumb: For 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.

Diluent focusing on injection Assay of propylgalate: mAU OH Chromolith Performance RP-18-e, 10 cm H O 300 x 4.6 mm ; 25 o C, 2 mL/min; O H O CH 3 250 Mobile phase: MeOH / ACN / aq. 0,1% O 200 H 3 PO 4 = 12 / 24 / 64 v/v/v; 150 V inj = 100  L; 2 100 [1] diluent = Mobile phase; [2] diluent = MeOH / ACN / aq. 0,1% 50 1 H 3 PO 4 = 20 / 40 / 40 v/v/v; 0 min 0 1 2 3 4 5

pH focusing on injection Assay of bromhexin: Br mAU CH 3 Hypersil BDS 3-C18, 10 cm x 4.6 C N H 2 mm x 3  m ; 25 o C, 1 mL/min; 800 Br NH 2 Mobile phase: ACN / aq. 0,1% 600 H 3 PO 4 at pH=7 with TEA : 75 / 25 v/v; 400 V inj = 5  L; 2 [1] diluent = Mobile phase; 200 [2] diluent = ACN / aq. 3 % HCl = 1 75 / 25 v/v; 0 min 0 5 10 15 20 25 30 35

Focusing due to the lack of the I.P. agent NH 2 mAU Assay of 2-aminopyridine: N 800 Purosphere Star-C18, 12.5 cm x 4.6 600 mm x 5  m ; 25 o C, 0.7 mL/min; Mobile phase: ACN / aq. 80 mM 400 1 Sodium Octane Sulfonate at pH 2.8 with H 3 PO 4 : 20 / 80 v/v; 200 V inj = 50  L; 2 0 [1] diluent = Mobile phase; 0 1 2 3 4 5 6 7 min 8 [2] diluent = ACN / aq. H 3 PO 4 at pH=2.8 : 20 / 80 v/v

1-Phenyl-2-butanol (k=1.25) Mobile phase Zorbax Eclipse XDB-C8 (150 mm x 4.6 mm x 5  m); Flow T o C = 15 o C;  S >  M.Ph. Mobile Phase: ACN / water : 60:40 (v/v); Flow rate: 1 mL/min; S – Mobile Phase V inj = 20  L Diluent (S) plunger S – ACN S – MeOH  Boundary 1:  gradient S = I-PrOH  Boundary 2:  gradient Fingering effect from. ref. [2] 2.4 2.6 2.8 3.0 3.2 3.4 [1] D. Cherrak et al., Viscous fingering: A systematic study of viscosity effects in methanol-isopropanol systems, Chromatographia , 46(11-12), 647-654 (1997). [2] S. Keunchkarian et al., Effect of sample solvent on the chromatographic peak shape of analytes eluted under RPLC conditions, J. Chromatogr. A , 1119, 20-28 (2006).

SF – t 0 k SF = (t R ) / t 0 RID resp. (mV) UV resp. (AU) 200.0 4.0 D A B C THF S = H 2 O D B C A 3.0 k SF S = aq. 50% THF S D i -PrOH B C A S 100.0 2.0 S = aq. 50% i -PrOH B C 1 A ACN S 1.0 D 1 C 2 D 2 S = aq. 50% ACN A D B C S MeOH S = aq. 50% MeOH 0.0 0.0 4.0 5.0 0.0 1.0 2.0 3.0 6.0 8.0 10.0 2.0 4.0 0.0 Time (min) Time (min) from ref. [3] A – Tartaric Acid; B – Malonic Acid; C – Acetic Acid; D – Maleic Acid. Column: YMC ODS-Aq C18 150 mm x 4.6 mm x 5  m; T o C = 35 o C; Flow rate = 1.25 mL/min; UV – 205 nm [3] E. Loesser et al., Using strong injection solvents with 100% aqueous mobile phase in RPLC, J. Sep. Sci. , 29, 2847-2852 (2006).

Column: Zorbax Eclipse C-8 150 mm x 4.6 mm x 5  m; OH mAU 100 Mobile Phase: ACN : aq. 0.1% H 3 PO 4 = 4/6 (v/v); 12 Flow Rate: 1 mL/min; Detection: UV – 291 nm; C(CH 3 ) 3 11 S = i -octane; V inj = 5 – 600  L 10 80 9 O 8 CH 3 60 7 k (BHA) 6 5 40 4 600  L 500  L 3 400  L 2 20 300  L 1 200  L 0 100  L 5  L min 0 100 200 300 400 500 600 700 0 0 10 20 V inj (  L) [4] V. David, C. Barcutean, C. Georgita, A. Medvedovici, Non-miscible solvent LVI-HPLC/DAD method for determination of butylated hydroxyanisole in lovastatin and simvastatin pharmaceutical formulations, Rev. Roum. Chim. , 5, 445-451 (2006).

Chromatographic Column Mobile Phase V M.Ph. A Analyte V S.Ph. V inj L S Diluent Stationary Phase

K A = k A x V M.Ph. / V S.Ph. k A = K A x V S.Ph. / V M.Ph. S is practically totally partitioned in the S.Ph. and exhibits similar properties; consequently: real = V S.Ph. + V inj V S.Ph. k A = K A x (V S.Ph. + V inj ) / V M.Ph. if V inj  than k A 

A (S)  A (M.Ph.) [1] A (M.Ph.) + L (S.Ph.)  A*L (S.Ph.) [2] K A = [A*L] S.Ph. / ([A] M.Ph. x [L] S.Ph. ) if assuming [S] >> [A] i S (M.Ph.) + L (S.Ph.)  S i *L (S.Ph.) K S = [S i *L] S.Ph. / ([S] i [3] M.Ph. x [L] S.Ph. ) A and K A =  1 x K ow A ; K S =  2 x K ow S > K ow S if assuming K ow than [3] ; [2] ; [1] ; [A*L] S.Ph. / [A] M.Ph. = k A x V M.Ph. / V S.Ph. ; k A = [(  1 x K ow A x [L] S.Ph. ) / V M.Ph. ] x V S.Ph. ; but a volume  V S.Ph. is available only for S;  V S.Ph. =  3 x V inj ; consequently  k A =  x (V S.Ph. –  V S.Ph. ) =  x V S.Ph. -  x  3 x V inj =  –  x V inj

  V      A S . Ph . k K L ; A 1 ow S . Ph . V M . Ph .            tot L L A L S L * * ; S . Ph . S . Ph . S . Ph . i S . Ph .         if assuming that A * L L S * L S . Ph . S . Ph . i S . Ph .  tot         n     tot tot L L L L S L L * ; ; S . Ph . S . Ph . i S . Ph . S Ph . . L V M S . Ph . w       V S   inj S S . Ph . S * L ;   i S . Ph . S i i M V w S . Ph .     V V         S inj A A L S . Ph . k K K ;    A 1 ow 1 ow L S M V i M V w M . Ph . w M . Ph .    k 0 L V M A      inj k 0 w S for (at ) q.e.d.! k 0 V V i ; A   A inj inj S M V w L S . Ph . [5] A. Medvedovici, Victor David, Vasile David, C. [5] A. Medvedovici, Victor David, Vasile David, C. Georgita, Retention phenomena induced by LVI of Georgita, Retention phenomena induced by LVI of solvents non-miscible with the mobile phase in RPLC, solvents non-miscible with the mobile phase in RPLC, J. Liq. Chromatogr. Relat. Technol. , 30, 199-213 J. Liq. Chromatogr. Relat. Technol. , 30, 199-213 (2007). (2007).

mAU 80 60 Single injection 400  L 40 1 0 2 3 4 5 6 7 8 9 10 11 12 13 14 15 4 x 100  L (48 min.) 20 1 x 400  L 0 min 0 2 4 6 8 10 12 4 x 100  L injections at 12 min. interval 1 2 3 6 0 4 5 7 8 9 10 11 12 13 14 15

Rule of five for LVI in diluents non-miscible with the M.Ph. 1 . D has increased chromatographic retention compared to target compounds (k SF > k A ); 2 . Solubility of D in the M.Ph. is low enough to force the saturation of the S.Ph. with D immediately after injection; 3 . Fingering effects due to different viscosities (D vs. M.Ph.) are controlled; 4 . D plug from a previous injection is already eliminated from the column before starting a new separation process; 5 . The initial chromatographic resolution supports the “apparent” reduction of the column length (affecting selectivity).

Solute: Metoprolol (log P = 1.88) Diluents: Methanol (log P = -0.77) Butyl acetate (log P = 1.78) H N Carbon tetrachloride (log P = 2.83) 1-Octanol (log P = 3.00) Cyclohexane (log P = 3.44) OH n-Hexane (log P = 3.9) Injection Volumes: 1, 5, 10, 20, 50, 75, 100  L O Chromatographic Columns: Zorbax XDB C-18 (150 mm x 4.6 mm x 5  m); Chromolith Performance C-18 (100 mm x 4.6 mm); C-18 Stable Bond AQ (150 mm x 4.6 mm x 5  m); Betasyl Phenyl (150 mm x 4.6 mm x 5  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)

V inj = 100  L mAU 8 7 6 5 n- C 6 4 CC 6 3 2 1-Octanol CCl 4 1 0 BuAc -1 MeOH M.Ph. 0 5 10 15 20 25 30 35 min

25 24 23 M.Ph. BuAc 22 CCl4 k 1-Octanol 21 CC6 n-C6 20 0 y = 0.0082x - 0.0669 R 2 = 0.8738 -0.01 19 Slope (Regress. k = f(V inj )) -0.02 18 0 20 40 60 80 100 120 -0.03 V inj -0.04 -0.05 -0.06 0.00 1.00 2.00 3.00 4.00 5.00 log P

14000 12000 10000 M.Ph. AcBu CCl4 8000 N 1-Octanol CC-6 6000 n-C6 4000 2000 5 0 4.5 0 20 40 60 80 100 120 V inj 4 3.5 M.Ph. 3 CCl4 AF 2.5 1-Octanol CC6 2 n-C6 1.5 1 0.5 0 0 20 40 60 80 100 120 V inj

30 y = -0.0185x + 14.115 y = -0.039x + 24.026 y = -0.0166x + 8.8189 R 2 = 0.9998 R 2 = 0.9999 R 2 = 0.9983 25 20 k 15 Phenyl C18 Chromolith C18 XDB 10 C18 AQ 5 y = -0.0136x + 12.818 R 2 = 0.9928 0 0 20 40 60 80 100 V inj

9000 Phenyl 8000 C18 Chromolith C18 XDB C18 AQ 7000 6000 5000 N 4000 3000 2000 1000 0 3 0 20 40 60 80 100 V inj Phenyl C18 Chromolith C18 XDB 2.5 C18 AQ 2 AF 1.5 1 0.5 0 0 20 40 60 80 100 V inj