O que é cromatografia líquida?

CROMATOGRAFIAHistórico

M. TSWEET (1903): Separação de misturas de

pigmentos vegetais em colunas recheadas com

adsorventes sólidos e solventes variados.

éter depetróleo

CaCO3

mistura depigmentos

pigmentosseparados

Cromatografia =

kroma [cor] + graph [escrever]

(grego)

•http://chemkeys.com/br/2000/07/18/cromatografia-a-gas-curso-em-diapositivos/

http://chemkeys.com/br/2000/07/18/cromatografia-a-gas-curso-em-diapositivos/

“Xpомофиллы в растительном и животном мире” (Chromophils in plant andanimal world) Doctor of Science dissertation,Warsaw, 1910, 380 pp. Reprinted fromChromatographic adsorption analysis, selected works of M. S. Tswet by Academyof Sciences of the USSR, 1946.

CROMATOGRAFIAPrincípio Básico

Separação de misturas por interação diferencial dos seus

componentes entre uma FASE ESTACIONÁRIA (líquido ou

sólido) e uma FASE MÓVEL (líquido ou gás).

CROMATOGRAFIAModalidades e Classificação

FM = Líquido

FM = Gás

Cromatografia

Líquida

Cromatografia

Gasosa (CG)

Em CG a FE

pode ser:

Sólida

Líquida

Cromatografia

Gás-Sólido (CGS)

Cromatografia

Gás-Líquido (CGL)

O equipamento

http://www.pharmainfo.net/reviews/introduction-analytical-method-development-pharmaceutical-formulations

O injetor

http://community.asdlib.org/imageandvideoexchangeforum/2013/08/02/loop-injector-for-hplc/

Parâmetros cromatográficos que você deve conhecer,

quando trabalha com cromatografia.

C.H. Collins, G.L. Braga, P.S. Bonato, Fundamentos de Cromatografia, 4ª edição .,:Editora da Unicamp, Campinas, 2006.

Cromatograma com as medidas relacionadas à determinação de parâmetros cromatográficos

M

R

M

MR

t

t

t

ttk

'

1

2

1

2

'

'

R

R

t

t

k

k

21

12

21

12 177,12hh

RR

bb

RRs

ww

tt

ww

ttR

Variação do volume de retenção (VM) da uracila e da tiouréia em função do tipo e da quantidade de modificador orgânico. Coluna: Restek Allure-C18 150 × 4,6 mm. VM = tM x F, onde F é a vazão da fase móvel Y. Kazakevich, R. Lobrutto, HPLC for Pharmaceutical Scientists, Wiley-InterscienceHoboken, 2005.

N

LH

22

545,516

h

R

b

R

w

t

w

tN

Medida e cálculo do fator de assimetria do pico. A. Braithwaite, F.J.Smith, “Chromatographic Methods”, 4a. edição, Chapman and Hall, Londres, 1985.

Medida e cálculo do fator de alargamento do pico, segundo a USP

Efeito da retenção, da eficiência e da seletividade sobre a resolução (adaptado da referencia

http://www.chromedia.org/chromedia?waxtrapp=yqegzCsHqnOxmOlIEcCbC&subNav=wnjedDsHqnOxmOlIEcCbCmF

CB

AH

Mt

L

R.M. Ornaf, M.W. Dong In S. Ahuja, M.W Dong (Eds) 6th Volume, Handbook of Pharmaceutical Analysis by HPLC, Elsevier Academic Press, Londres, 2005, pg 19-45.

Eddy difusio

a) Gráficos de van Deemter b) gráficos de Knox . Dm para a acetofenona é 1.2 × 10−9

m2/s . Fase móvel 30/70 acetonitrile/água temperatura: 40 ◦C, Colunas Acquity BEH

C18, 1.7 m, 10 cm × 2.1mm ID; XBridge C18, 3.5m, 15 cm×4.6mm ID; XBridge C18,

5 m, 25 cm×4.6mm ID. de Villiers et al. / J. Chromatogr. A 1127 (2006) 60–69

a)

b)

𝐻 = 𝐴𝑑𝑝 +𝐵𝐷𝑀

𝜇+ 𝐶

𝑑𝑝 2𝜇

𝐷𝑀 𝑣 =

𝜇𝑑𝑝

𝐷𝑚 ℎ =

𝐻

𝑑𝑝 ℎ = 𝑎 +

𝑏

𝑣+ 𝑐𝑣 1

Fekete et al., Journal ofChromatography A 1228 (2012) 57-71

ΔP = (ηFL)/(K0πr2dp2)onde K0 é a permeabilidade específica, ηé a viscosidade da FM, F é a vazão da FM,r é o raio interno da coluna e dp é odiâmetro médio das partículas da faseestacionária (FE) que recheiam a coluna

Quais são os principais modos de cromatografia?

Different mechanisms of retardation in liquid chromatography (J.F. Rubinson, K.A.Rubinson, Contemporary ChemicalAnalysis, Prentice Hall, Upper SaddleRiver, 1998).

http://www.chromedia.org/chromedia?waxtrapp=yqegzCsHqnOxmOlIEcCbC&subNav=yarwnEsHqnOxmOlIEcCzBYT

Normal-phase LC

NP-LC is performed by using silica sorbents which or chemically

modified with polar and/or hydrophilic functional groups and in

combination with non-polar eluents.

http://www.expertsmind.com/topic/packing-material-or-stationary-phase/adsorption-chromatography-913002.aspx

Typical chromatogram of a reaction mixture collected during the course of reaction of phthalicanhydride with benzene in the presence of AlCl3, as catalyst. Peaks: 1, benzene; 2, anthraquinone; 3, phthalic anhydride; 4, maleic anhydride; 5, unknown. Chromatographicconditions: Column: Spherisob silica, 250 × 4.6mm, 10μm; mobile phase, n-heptane–ethanol chloroform–acetic acid (89 : 5 : 5 : 1, v/v/v/v); flow rate, 1mL/ min; detection, UV at 254 nm; temperature, 27°C.

Reversed phase chromatography

Viscosity as a function of organic/water composition

(A) 30% MeCN: 70% 20mM Phosphate, pH 7. (B) 50% MeCN: 50% 20mM Phosphate, pH 7. (C) 80% MeCN: 20% 20mM Phosphate, pH 7.

Retention of alkylbenzenes on Luna-C18 column from acetonitrile/watereluent.

%ACN tR(NB), min k(NB) tR(PP), min k(PP) α (PP/NB)

100 1,02 0,28 1,02 0,28 1 90 1,04 0,3 1,04 0,3 1 80 1,18 0,48 1,12 0,39 0,83 70 1,38 0,73 1,27 0,59 0,81 60 1,73 1,16 1,57 0,96 0,83 50 2,37 1,96 2,29 1,86 0,95 40 3,73 3,66 3,73 3,66 1 30 6,55 7,19 8,62 9,78 1,36 25 9,25 10,56 15,35 18,19 1,72 20 13,46 15,83 30,75 37,44 2,37 %MeOH tR(NB),min k(NB) tR(PP), min k(PP) α (PP/NB)

100 1,02 0,28 1,02 0,28 1 90 1,08 0,35 1,08 0,35 1 80 1,25 0,56 1,25 0,56 1 70 1,5 0,88 1,68 1,1 1,26 60 2,02 1,53 2,73 2,41 1,58 50 3,05 2,81 5,65 6,06 2,16 40 5,07 5,34 14,36 16,95 3,18 30 8,91 10,14 41 50,25 4,96 25 11,78 13,73 74 91,5 6,67

Tempo de retenção (tR), fator deretenção (k) e seletividade (α) donitrobenzeno (NB) e dopropilparabeno (PP) em funçãoda percentagem e do tipo demodificador orgânico. Coluna:Symmetry C18, 3 μm, 75 × 4,6mm, Waters. Vazão: 1mL/min.Temperatura 40 °C

Y. Kazakevich, R. Lobrutto, HPLC for Pharmaceutical Scientists, Wiley-Interscience Hoboken, 2005.

Selectivity for steroids as a function of organic mobile-phasecomponent. Chromatograms showing the elution order of all eightcongeners as a function of organic modifier with 0.1% formic acid asthe buffer phase on YMC ODS-AQ column at ambient temperature.(Top panel) 25% acetonitrile, 1.5-mL/min flow rate; (middle panel)45% methanol, 1.2-mL/min flow rate; (bottom panel) 20%tetrahydrofuran, 1.5- mL/min flow rate. (Reprinted from reference51, with permission.) P. Zhuang, R. Thompson, and T. O’Brien, J. Liq.Chrom. Rel. Technol. 28 (2005), 1345–1356.

Time (min) A (%) B (%)0 100 04 55 4540 0 10045 0 10048 100 0

A. Stafiej et al. / J. Biochem. Biophys. Methods 69 (2006) 15–24

Y. Kazakevich, R. Lobrutto, HPLC for Pharmaceutical Scientists, Wiley-Interscience Hoboken, 2005.

Theoretical retention versus pH profiles for acidic and a zwitterionic component. Chromatographicconditions: Column: 15-cm × 0.46-cm Phenoemenex Luna C18(2), 5μm; 70% 15mM K2HPO4 adjusted pH 2–9 with phosphoric acid; 30% MeCN; flow rate, 1mL/min; temperature, 25°C. R. LoBrutto, A. Jones, Y. V. Kazakevich, and H. M. McNair, J. Chromatogr. A 913 (2001), 173–187

Y. Kazakevich, R. Lobrutto, HPLC for Pharmaceutical Scientists, Wiley-Interscience Hoboken, 2005.

R. LoBrutto, A. Jones, Y. V. Kazakevich, and H. M. McNair, J. Chromatogr. A 913 (2001), 173–187

Y. Kazakevich, R. Lobrutto, HPLC for Pharmaceutical Scientists, Wiley-Interscience Hoboken,

2005.

http://pubs.acs.org/cen/coverstory/86/8617cover.html acessada em 2 de fevereiro de 2011.

Dolan, J. W.; LCGC North Am. 2007, 25, 1014

Diferentes seletividades obtidas para uma mistura de compostos básicos com fases estacionárias preparadas com

diferentes tipos de ligantes químicos sobre a mesma sílica. Colunas: 250 x 4,6 mm, dp 5 μm, todas da ACE. Fase

móvel: metanol:tampão fosfato (pH 6; 25 mmol/L) 80:20 (v/v). Vazão: 1.5 mL/min. Identificação dos solutos: 1 =

norefedrina; 2 = nortriptilina; 3 = tolueno; 4 = amitriptilina; 5 imipramina

J. Layne / J. Chromatogr. A 957 (2002) 149–164

Separation of tricyclic antidepressants on an (a) Prontosil C18 H and (b) Prontosil C 18 ACE/EPS column (150 mm × 4.6 mm, dp 5 μm). Mobile phase 65:35 (v/v) methanol:phosphate buffer (20 mM, pH 7). Peaks: 1 = uracil, 2 = protriptyline, 3 = nortriptyline, 4 = doxepine, 5 = imipramine, 6 = amitryptyline, 7 = trimipramine, 8 = clomipramine.

H. Engelhardt, R. Grüner, M. Schererv

Chromatographia, 53 (2001), pp. S154–S161

O que acontece quando usamos partículas com

diâmetro reduzido?

R.M. Ornaf, M.W. Dong In S. Ahuja, M.W Dong (Eds) 6th Volume, Handbook of Pharmaceutical Analysis by HPLC, Elsevier Academic Press, Londres, 2005, pg 19-45.

Experimental H–u plots of columns packed with 1.3, 1.7, 2.6 and 5 μm core–shell particles (peak widths were corrected for the extra-column band broadening). Test analyte: butylparaben.

S. Fekete, D. Guillarme / J. Chromatogr. A 1308 (2013) 104– 113

Impurity profiling of α-estradiol. Peaks: α-estradiol (1), main impurity (2) and minor impurity (3). Columns: Phenomenex Kinetex 50 mm × 2.1 mm, 1.3, 1.7 and 2.6 μm. Mobile phase: water/ACN 60/40 (v/v), flow rate: 0.5 mL/min, temperature: 25 °C, Vinj: 0.5 μL (25 μg/mL estradiol), λ = 210 nm (80 Hz).

S. Fekete, D. Guillarme / J. Chromatogr. A 1308 (2013) 104– 113

Fast separation of cashew nut extract. Columns: Phenomenex Kinetex 50 mm × 2.1 mm, 1.3, 1.7 and 2.6 μm. Mobile phase: water/ACN 14/86 (v/v), flow rate: 0.8 mL/min, temperature: 25 °C, Vinj: 0.3 μL, λ = 280 nm (80 Hz).

S. Fekete, D. Guillarme / J. Chromatogr. A 1308 (2013) 104– 113

Transferência de método do HPLC para o UPLC, metodologia empregada para analisar uma formulação

farmacêutica de composto 6 onde se encontram onze impurezas (a) método original para HPLC: coluna,

XBridge C18 (150×4.6 mm, 5 μm); vazão: 1000 μL min−1; volume de injeção, 20 μL; tempo de gradiente, 45

min. (b) método UHPLC transferido para HPLC: coluna, Acquity BEH C18 (50×2.1 mm, 1.7 μm); vazão,

610 μL min−1; volume de injeção, 1.4 μL; tempo de gradiente, 5.1 min. (c) Otimização do método UHPLC:

columa, Acquity BEH C18 (50×2.1 mm, 1.7 μm); vazão, 1000 μL min−1; volume de injeção, 1.4 μL; tempo

total de gradiente, 3.1 min. Figura adaptada Guillarme et al. Anal Bioanal Chem (2010) 397:1069–1082.

Separation of a pharmaceuticalformulation “Rapidocain” in isocratic mode. Experimental conditions: mobile phasecontaining ACN/phosphate buffer at pH 7.2 (40:60, v/v). A. Waters Xterra RP18, 150 × 4.6 mm, 5 μm, F = 1000 μl/min,Vinj = 20 μl. B. Waters Acquity Shield RP18, 50 × 2.1 mm, 1.7 μm, F = 613 μl/min, Vinj = 1.4 μl. C. Waters Acquity Shield RP18, 50 × 2.1 mm, 1.7 μm, F = 1000 μl/min, Vinj = 1.4 μl. 1, methylparaben; 2, 2,6-dimethylanalinine; 3, propylparaben; 4, lidocaine.(http://www.rsc.org/shop/books/2012/9781849733885.asp).

S. Fekete et al. / Journal of Pharmaceutical and Biomedical Analysis 87 (2014) 105–119

Chromatograms for columns with different lengths. Conditions: 50, 100, and 150 mm BEH Shield C-18 columns packed with 1.7, 3.5, and 5.0 μmparticles respectively; 2.1 mm internal diameter; flow rates for 50, 100, and 150 mm lengths were 0.36, 0.24, and 0.12 mL/min, respectively; the injection volumes for 50, 100, and 150 mm lengths were 1.0, 2.0, and 3 μL, respectively; the sample concentration was 0.1 mg/mL for each analyte;. Peak identifications: from left to right (1) uracil; (2) benzylalcohol; (3) acetophenone; (4) propiophenone; and (5) benzophenone

N. Wu, A.C. Bradley / J. Chromatogr. A 1261 (2012) 113–120

Chromatograms for columns withdifferent lengths. Conditions: 50,100, and 150 mm BEH Shield C-18columns packed with 1.7, 3.5, and5.0 μm particles respectively;2.1 mm internal diameter; flowrates for 50, 100, and 150 mmlengths were 0.36, 0.24, and0.12 mL/min, respectively; theinjection volumes for 50, 100, and150 mm lengths were 1.0, 2.0, and3 μL, respectively; the sampleconcentration was 0.1 mg/mL foreach analyte;. Peak identifications:from left to right (1) uracil; (2)benzylalcohol; (3) acetophenone;(4) propiophenone; and (5)benzophenone.

Loss in efficiency at various retention factors for columns with differentlengths. Conditions: 0.1 mg/mL uracil, benzylalcohol, acetophenone, propiophenone; and benzophenone were used as analytes. Keys: (♦) 50 mm; (○) 100 mm; and (●) 150 mm.

Chromatograms obtained with a Kinetex® 100 × 2.1 mm, 2.6 μmcolumn with A. a conventional HPLC system without modification, and B. after optimization. https://phenomenex.blob.core.windows.net/documents/ad6882fd-5ad1-478a-ab0a-5ad746734529.pdf (06.02.2013).

Graphical representation of A. pressure tolerance and type of pumping system, and B. standardsystem dwell volume of all the UHPLC systems (ΔP > 600 bar) commercially available. In A., lightred expresses low pressure system volume while dark blue represents high pressure systemvolume. It is important to notice that the standard dwell volume reported in this figure can bemodified on a few instruments either by bypassing the damper and mixer, or by changing thevolume of the mixing chamber. (For interpretation of the references to colour in this figurelegend, the reader is referred to the web version of this article.)

Jul 1, 2012By: Fabrice Gritti, Georges GuiochonLCGC North America Volume 7, Issue 30

Distribuição do tamanho de

partícula. (A) Zorbax-XDB-

1.8 μm, (B) Acquity-BEH-

1.7 μm, (C) Kinetex-1.7

μm, and (D) EiS-150-1.7

μm. (E) Sobreposição de

todas as figuras.

Omamogho & Glennon;

Anal. Chem. 2011, 83,

1547-1556

DA,B é o coeficiente de difusão de um soluto A em um solvente

B, (cm2 s−1), MB é a massa molecular do solvente B (g mol−1),

T é a temperatura absoluta em (K), ηB é a viscosidade do

solvente B (cP) a uma temperatura T, VA é o volume molar do

soluto A (cm3 g−1 mol−1) e ϕB, é o coeficiente de associação do

soluto com a fase móvel B (adimensional) (Guillarme et al /

Journal of Chromatography A, 1052 (2004) 39–51)

Curvas H–u (curvas de van Deenter) de colunas 1.7 µm core–shell (Kinetex C18, 5 cm×2.1mm) e 1.7 µm

totalmente porosas (Waters BEH C18, 5 cm×2.1mm). Fase móvel: 440/560/1 (para proteínas de 18.8 kDa),

470/530/1 (para proteínas de 38.9 kDa) e 610/390/1 (para BSA, 66.3 kDa) acetonitrila/água/TFA,

temperatura: 60 ◦C, injeção: 0.5 µl, analitos teste: proteínas. S. Fekete et al. / Journal of Pharmaceutical and

Biomedical Analysis 54 (2011) 482–490.

Van Deemter plots das colunas Kinetex, Ascentis Express (2.7 μm), e sub-2 μm totalmente porosas. Fase

móvel : (A) 48% acetonitrila–52% água para o estradiol e (B) 95% acetonitrila–5% água para a ivermectina.

Temperatura: 35 °C, injeção: 0.5 μL, analito: (A) estradiol (B) ivermectina. (E. Olah et al. / J. Chromatogr. A

1217 (2010) 3642–3653)

Waters Acquity BEH 50 × 2.1 mm, 1.7 μm, C18 column. The mobile phase containing a mixture of 0.01% aqueous trifluroaceticacid and acetonitrile in the ratio of 75:25 (v/v) at a flow rate of 500 μl/min was used

A Kromasil C18, 250 × 4.6 mm, 5 μmcolumn was used for separation. Chromatographic separation was achieved in both the modes (isocratic and gradient). Mobile phase consisting of a mixture of A: 0.01% aqueous trifluroacetic acid and B: acetonitrile in the ratio 75:25 (v/v) for isocratic mode

Journal of Pharmaceutical and Biomedical Analysis 46 (2008) 236–242

Chemical structures of primaquine phosphateand impurities. (A) primaquine phosphate (B) impurity I: 8-(4-amino-4-methylbutyl amino)-6 methoxyquinoline (C) impurity II: 8-nitro-6-methoxyquinoline.

The LOD and LOQ demonstrated for HPLC (isocratic mode) and UPLC.

O efeito da pressão na retenção.

Effect of pressure (generatedby restrictor tubes at columnoutlet) on the retention ofsmall analytes. Column: AcquityBEHC18 (50 mm × 2.1 mm), mobile phase: water (0.1% TFA) + acetonitrile (0.1% TFA): 70 + 30 (v/v), flow-rate: 100 μL/min, temperature: 30 °C, injected volume: 0.5 μL, detection: 210 nm. Peaks: lidocaine (1), salicylic acid (2), bupivacaine (3), propranolol (4), propylparaben (5) andtestosterone (6).

S. Fekete et al. / J. Chromatogr. A 1270 (2012) 127– 138

Effect of pressure (generated byrestrictor tubes at column outlet) onthe retention of related peptides(1–1.3 kDa). Column: AcquityBEH300 C18 (50 mm × 2.1 mm), mobile phase: water (0.1% TFA) + acetonitrile (0.1% TFA): 73 + 27 (v/v), flow-rate: 100 μL/min, temperature: 40 °C, injectedvolume: 1 μL, detection: fluorescence ex.: 280 nm, em.: 360 nm. Peaks: P-868 (1), P-866 (2), P-870 (3), and P-869 (4).

Effect of pressure (generated by restrictor tubes at column outlet) on the retention of insulin and related proteins. Column: Acquity BEH300 C18 (50 mm × 2.1 mm), mobile phase: water (0.1% TFA) + acetonitrile (0.1% TFA): 69 + 31 (v/v), flow-rate: 100 μL/min, temperature: 50 °C, injected volume: 1 μL, detection: fluorescence ex.: 280 nm, em.: 360 nm. Peaks: insulin (1), related proteins (2–4). Please note that time scale is normalized to the last eluted peak.

Effect of pressure (A) and thecombination of pressure andmobile phase velocitiy (B) onthe retention (relativechange in k) of different sizeanalytes (1.1 kDa peptide, 5.7 kDa insulin, 12.3 kDacytochrome C and 17 kDamyoglobin). Column: AcquityBEH300 C18 (50 mm × 2.1 mm), mobilephase A: water (0.1% TFA), mobile phase B: acetonitrile(0.1% TFA). Detection: fluorescence ex.: 280 nm, em.: 360 nm. Investigatedpressure range: 100–1100 bar.

Effect of very highpressure (generated byrestrictor tubes atcolumn outlet) on theretention (A) and peakshape (A and B) ofcytochrome C. Column: Acquity BEH300 C18 (50 mm × 2.1 mm), mobile phase: water(0.1% TFA) + acetonitrile(0.1% TFA): 70.5 + 29.5 (v/v), flow-rate: 200 μL/min, temperature: 65 °C, injected volume: 1 μL, detection: fluorescenceex.: 280 nm, em.: 360 nm.

M.M. Fallas et al. / J. Chromatogr. A 1297 (2013) 37– 45

Effect of pressure onselectivity of separation of a diverse mixture ofcompounds Column: AceExcel 5 C18, 5 cm × 0.21 cm I.D., 5 μm, mobile phase 25% ACN in 0.025 M phosphatebuffer at pH 2.7. T = 30 °C, F = 0.3 mL/min, λ = 254 nm. Peak identities 1 = uracil, 2 = aniline, 3 = 2-naphthalenesulfonic acid, 4 = propranolol, 5 = prednisone, 6 = acetophenone, 7 = diphenhydramine, 8 = nitrobenzene. Results for this Figure obtained with theAcquity system.

Análise de Macro-moléculas

Wide-pore fused-core particles for proteinseparations. Left: cartoon of particle dimensions.Right: scanning electron micrograph of particles.

S.A. Schuster et al. / J. Chromatogr. A 1315 (2013) 118– 126

Focused ion beam scanning electron micrographs of wide-pore fused-core particles. Left: single particle showing porous outer shell. Right: particle cross-section showing shell thickness.

S.A. Schuster et al. / J. Chromatogr. A 1315 (2013) 118– 126

Plate height comparison. Columns 100 mm × 2.1 mm; mobile phase: 1.7 μm totally porous –40.5% acetonitrile/59.5% aqueous 0.1% (v/v) trifluoroacetic acid, = 3.4; 3.4 μm Halo 400 –42.5% acetonitrile/57.5% aqueous 0.1% (v/v) trifluoroacetic acid, k = 3.6; solute: carbonicanhydrase (29 kDa), 0.1 mg/mL in 6 M urea/1.0% acetic acid; temperature: 60 °C

Reduced plate height comparison. Columns 100 mm × 2.1 mm; mobile phase: 1.7 μm totallyporous – 40.5% acetonitrile/59.5% aqueous 0.1% (v/v) trifluoroacetic acid, = 3.4; 3.4 μm Halo 400 – 42.5% acetonitrile/57.5% aqueous 0.1% (v/v) trifluoroacetic acid, k = 3.6; solute: carbonicanhydrase (29 kDa), 0.1 mg/mL in 6 M urea/1.0% acetic acid; temperature: 60 °C

Effect of temperature on protein separations. Column: 100 mm × 2.1 mm Halo Protein C4; gradient: 28–58% B in 10.0 min; mobile phase A: aqueous 0.1% trifluoroacetic acid; mobilephase B: acetonitrile with 0.1% trifluoroacetic acid; flow rate: 0.45 mL/min; instrument: Agilent1200 SL; injection volume, 2 μL; detection: 215 nm; temperatures as indicated; solutes, proteinsas shown.

Effect of pore size on separation of small proteins. Columns: 100 mm×4.6 mm Halo C18 (90 Å pores) and 100 mm×4.6 mm Halo Peptide ES-C18 (160 Å pores); mobile phase: A=water/0.1% trifluoroacetic acid; B=acetonitrile/0.1% trifluoroacetic acid; gradient: 25–42% B in 10 min; flowrate: 1.5 mL/min; temperature: 30 °C; detection: 215 nm; and solutes in order of elution: (1) ribonuclease A, (2) bovine insulin, (3) human insulin, (4) cytochrome c, and (5) lysozyme. Peakwidths in minutes above each peak. Journal of Pharmaceutical Analysis 2013;3(5):303–312

Fases estacionárias monolíticas

SEM photographs of monolithic silica rod columns prepared at different PEG concentrations in the startingreaction mixture. PEG: 9.4 g (a), 9.8 g (b), 10.2 g (c), and 10.4 g (d). Other starting reaction conditions:45 mL TMOS, 100 mL of 0.01 M aqueous acetic acid solution. Concentration of NH4OH used for controllingthe mesopore size: 0.01 M. The skeleton size and through-pore size are indicated by black and whitearrows, respectively, in (a). Journal of Chromatography A, 1191 (2008) 231–252

Experimental Van Deemterplots of 2.6 μm shell-type(Kinetex), 2.7 μm shell-type(Ascentis Express), sub-2 μm totally porousparticles and a monolithcolumn (peak widths werecorrected for the extra-column broadening).Mobile phase: (A) 48%acetonitrile–52% water forestradiol and (B) 95%acetonitrile–5% water forivermectin, temperature:35 °C, injection: 0.5 μL,analyte: (A) estradiol and(B) ivermectin. Journal of

Chromatography A,

Volume 1217, Issue 23,

2010, 3642 - 3653

Structures and possible fragmentations of each analyte and IS.

Yin Huang , Yuan Tian , Zunjian Zhang , Can Peng Journal of Chromatography B,

Volume 905, 2012, 37 - 42

ZIC®-HILIC column (250 mm × 4.6 mm, 5 μm) from Merck (Darmstadt, Germany).The column temperature was maintained at 45 °C with an injection of 10 μL. Mobile phase A consisted of acetonitrile while mobile phase B onsisted of 10 mM ammonium acetate in redistilled water. The isocratic program was 70% A and 30% B at a flow rate of 0.5 mL/min. Y. Huang et al. / J. Chromatogr. B 905 (2012) 37–42.

Effect of water content on the retention

of hydrazines on a ZIC HILIC column.

Column temperature was at 30 °C with a

flow rate of 0.4 mL/min using a splitter

and CLND. The CLND system was set at

1050 °C combustion furnace, 50 mL/min

argon, 280 mL/min oxygen, 75 mL/min

makeup (argon), 30 mL/min ozone, 5 °C

cooler, gain x1, 750 V on PMT. The

analyte concentrations were about 30–

70 μg/mL in water/EtOH (20/80, v/v).

Acetonitrile at 0.1% (v/v) level in EtOH

was used as a void time marker. Injection

volume was 10 μL. (A) Chromatographic

separation of hydrazines as a function of

water content in the mobile phase–

TFA/water/ethyl alcohol (0.1/50–10/50–

90, v/v/v). 1: 1,2-Dimethylhydrazine, 2:

1,1-dimethylhydrazine, 3:

methylhydrazine and 4: hydrazine. (B)

Effect of water content on k′ in the

isocratic condition with the mobile

phases–TFA/water/ethyl alcohol (0.1/50–

10/50–90, v/v/v). Hydrazine (green),

methylhydrazine (red), 1,1-

dimethylhydrazine (pink) and 1,2-

dimethylhydrazine (blue).

Chemical structures of SBD-F and thiols. (A) Structure of thiols: 1, Cys; 2, Hcy; 3, CA; 4, γ-GluCys; 5, CysGly; 6, GSH; 7, NAC; 8, MPG. (B) Reaction of SBD-F and thiolsAnalyst,2013, 138, 3802-3808.

Effect of the type of alcohols on the retention of hydrazines on a ZIC HILIC column. For eachseparation, isocratic run was performed in the mobile phase of TFA/water/alcohol (0.1/10/90,v/v/v), column temperature at 30 °C, flow rate at 0.4 mL/min with a splitter and CLND. 1: 1,2-Dimethylhydrazine, 2: 1,1-dimethylhydrazine, 3: methylhydrazine and 4: hydrazine.

Effect of the type of acid modifiers on the retention of hydrazines on a ZIC HILIC column. Foreach separation, isocratic elution was performed with acid/water/ethyl alcohol (0.1/30/70,v/v/v) as a mobile phase, column temperature at 30 °C, flow rate at 0.4 mL/min with a splitterand CLND. 1: 1,2-Dimethylhydrazine, 2: 1,1-dimethylhydrazine, 3: methylhydrazine and 4:hydrazine.

The separation of hydrazines on different columns. Column temperature was at 30 °C; flow rate was 0.4 mL/min with a splitter and CLND. Mobile phase was formic acid/water/ethyl alcohol(0.5/20/80, v/v/v). 0.1% ACN (v/v) in ethyl alcohol was used as a void volume marker. 1: 1,2-Dimethylhydrazine, 2: 1,1-dimethylhydrazine, 3: methylhydrazine and 4: hydrazine. (a) ZorbaxNH2, (b) Diol, (c) Amide-80 and (d) ZIC HILIC.

HILIC columns Functional group Chemical structure

Inertsil SIL Bare silica

Inertsil Amide Amide

Inertsil Diol Diol

TSKgel NH2-100 Amino

PC HILIC Phosphorylcholine

ZIC-HILIC Sulfobetaine

Retention time of

SBD–thiols on

five HILIC columns.

Columns: Inertsil Diol,

●; Inertsil SIL, ◇;

PCHILIC, ▲;

Inertsil Amide, ; ZIC-

HILIC, ◆. Column

temperature: 35

°C. Mobile

phase: acetonitrile–10

mmol l−1 ammonium

formate buffer (pH 3.0)

= 75/25 (v/v). Linear

velocity: 58 mm

min−1. Fluorescence

detection: ex; 375 nm,

em; 510 nm.

Effect of (A) acetonitrile content,

(B) pH of ammonium

formate buffer and (C)

concentration ofammonium

formate buffer on retention time

of SBD–thiols. Symbols: SBD–

MPG, ●; SBD–NAC,◇; SBD–

CA, ▲; SBD–Hcy, ; SBD–Cys,

◆; SBD–CysGly, ○; SBD–GSH,

■; SBD–γ-GluCys, △. Column:

ZIC-HILIC (150 mm × 2.1 mm

i.d., 5 μm, Merck). Column

temperature: 35 °C. Mobile phase:

(A) acetonitrile–10 mmol

l−1 ammonium formate buffer (pH

3.0), (B) acetonitrile–10 mmol

l−1 ammonium formate buffer =

75/25 (v/v), and (C) acetonitrile–

ammonium formate buffer (pH

3.0) = 75/25. Flow rate: 0.2 ml

min−1. Fluorescence detection: ex

375 nm, em 510 nm. Injection

sample: 5 μl, containing

90%acetonitrile.

Comparison of five HILIC columns: amide, hybrid silica, diol, bare silica and zwitterionic phase,

for the separation of a test mixture of acidic compounds: (AT) coumachlor, (BJ)

hydrochlorothiazide, (BP) ketoprofen, (AY) diclofenac, (AD) acetylsalicylic acid, (BH)

furosemide. Column dimensions: 2.1 mm × 50 mm, 1.7 μm, except for RRHD 1.8 μm. Flow rate

of 0.5 mL/min, λ = 230 nm, injected volume = 1 μL. Condition: ammonium formate (50 mmol/L,

pH 4), gradient profile: 95% ACN for 1 min, then 95–65% ACN in 3 min with T = 30 °C.

Comparison of five HILIC columns: amide, hybrid silica, diol, bare silica and zwitterionic phase, for the

separation of a mixture of basic compounds: (CJ) noscapine, (AF) alprazolam, (BI) heroin, (CV) sotalol,

(AS) codeine, (BK) hydromorphone, (AZ) dihydrocodeine. Column dimensions: 2.1 mm × 50 mm,

1.7 μm, except for RRHD 1.8 μm. Flow rate of 0.5 mL/min, λ = 249 nm, injected volume = 1 μL.

Condition: ammonium formate (50 mmol/L, pH 4), gradient profile: 95% ACN for 1 min, then 95–65%

ACN in 3 min with T = 30 °C.

Effect of pH on selectivity.Column: bare silica(2.1 mm × 50 mm,1.8 μm), flow rate of0.5 mL/min., λ = 249 nm,injected volume = 1 μL.Condition: pH 3ammonium formate50 mmol/L, pH 4ammonium formate50 mmol/L, pH 5ammonium acetate50 mmol/L, pH 6ammonium acetate50 mmol/L, gradientprofile: 95% ACN for1 min, then 95–65% ACNin 3 min withT = 30 °C.Peak label: (CL) oxazepam,(AY) diclofenac, (BQ)lidocaine, (CP) pindolol,(CZ) thebaine, (BK)hydromorphone, (AZ)dihydrocodeine

Effect of organic modifier onselectivity: ACN, ACN/IPA 80:20v/v, and ACN/MeOH 80:20 v/v.Column: silica bare, flow rate of0.5 mL/min, λ = 249 nm,injected volume = 1 μL.Condition: ammonium formate(50 mmol/L, pH 4), gradientprofile: 5% buffer for 1 min,then 5–35% buffer in 3 minwith T = 30 °C. Peak label: (CL)oxazepam, (AY) diclofenac, (BQ)lidocaine, (CP) pindolol, (CZ)thebaine, (BK) hydromorphone,(AZ) dihydrocodeine.

Performance comparison of two HILIC columns: (A) an Acquity BEH HILIC (2.1 mm id × 150 mm,1.7 μm) and (B) an Acquity HILIC amide (2.1 mm id × 150 mm, 1.7 μm) for the analysis of amixture of hypoxanthine (1: 80 μg/mL), cytosine (2: 10 μg/mL), nicotinic acid (3: 30 μg/mL) andprocainamide (4: 30 μg/mL) dissolved in pure ACN. Conditions: mobile phase: ammoniumformate (50 mM, pH 3.14) modified with ACN, gradient profile: 95% ACN for 6 min, then 95–75% ACN in 5 min for (A) and isocratic conditions: 94% ACN for (B), flow rate of500 μL/min, λ = 214 nm, volume injected = 5 μL, T = 30 °C.

Comparison of the chromatographic performance obtained by (A) RPLC vs. (B) HILIC for the analysis of a mixture of 9 peptides: (1) lysine vasopressin (20 μg/mL), (2) arginine vasopressin (12 μg/mL), (3) peptide D (20 μg/mL), (4) triptorelin (5 μg/mL), (5) peptide A (20 μg/mL), (6) insulin (60 μg/mL), (7) peptide B (6 μg/mL), (8) peptide E (25 μg/mL), (9) peptide C (6 μg/mL) dissolved in water for (A) and in IPA for (B); thestar (*) designates an impurity present in synthetic peptides. Conditions: (A) Column Acquity BEH C18 (2.1 mm id × 150 mm, 1.7 μm), flow rate of 400 μL/min, λ = 214 nm, volume injected = 5 μL, gradient profile: 10–90% ACN in 20 min with T = 30 °C, (B) Column Acquity HILIC amide (2.1 mm id × 150 mm, 1.7 μm), flowrate of 500 μL/min, λ = 214 nm, volume injected = 5 μL, gradient profile: 90% ACN for 3 min, then 90–62% ACN in 9 min with T = 30 °C.

size exclusion chromatography

Illustrative description of separation in SEC

HONG, Paula; KOZA, Stephan; BOUVIER, Edouard SP. A REVIEW SIZE-

EXCLUSION CHROMATOGRAPHY FOR THE ANALYSIS OF PROTEIN

BIOTHERAPEUTICS AND THEIR AGGREGATES. Journal of liquid

chromatography & related technologies, v. 35, n. 20, p. 2923-2950, 2012.

FEKETE, Szabolcs et al. Theory and practice of Size Exclusion

Chromatography for the analysis of protein aggregates. Journal of

Pharmaceutical and Biomedical Analysis, 2014.

The free energy change of a chromatographic process can be described by, where ΔG 0, ΔH 0,and ΔS 0 are the standard free energy, enthalpy, and entropy differences, respectively; R is thegas constant: T is absolute temperature, and k is the partition coefficient. For mostchromatographic modes of separation, the enthalpy of adsorption is the dominant contributorto the overall change in free energy. SEC is unique in that partitioning is driven entirely byentropic processes as there ideally is no adsorption, ΔH = 0. Thus the previous equationbecomes: where K D is the thermodynamic retention factor in SEC. Thus, in SEC separations,temperature should have no impact on retention. In practice, temperature can indirectly impactretention to a small degree by altering the conformation of the proteins, as well as by affectingmobile phase viscosity and analyte diffusivity.

The thermodynamic SEC retention factor is the fraction of intraparticle porevolume that is accessible to the analyte: where V R , V 0, and V i are the respectiveretention volumes of the analyte of interest, the interstitial volume, and the intra-particle volume. K D will range from a value of 0 where the analyte is fully excludedfrom the pores of the stationary phase, to a value of 1 where the analyte fullyaccesses the intraparticle pores.

Separation of (1) thyroglobulin, (2) IgG, (3) BSA, (4) Myoglobin, and (5) Uracil on a Waters ACQUITY UPLC BEH200 SEC, 1.7 µ, 4.6 × 150 mm. Mobile phase: 100 mM sodium phosphate, pH 6.8. Flow rate: 0.3 mL/min. Temperature: 30°C (black), 40°C (blue), 50°C (red). Reproducedwith permission from Waters Corporation, Milford, MA

Typical SEC calibration curve

Theoretically expected impact of the particle size and mobile phase temperature on column performance. (For the calculations, a 50 kDa protein was assumed.).

Effect of linear velocity on plate height for (a) ribonuclease A (red) and (b) a monoclonalantibody (blue) on two columns varying in particle size. The 4.6 × 150 mm columns were packedwith either 1.7 micron (solid line) or 2.6 micron particles (dashed line). Pore size of stationaryphase sorbent: 200 Å. Mobile phase consisted of 100 mM sodium phosphate, pH 6.8.Reproduced with permission from Waters Corporation, Milford, MA.

Comparison of Columns: Effect of Particle Size on Efficiency and Resolution for a Reduced Antibody

Theoretical Plates

[-17pt] Columns

Dimensions (m i.d. ×

mm length)Particle

size (μm)Pore sizes

(Å) HC LC Resolution

TSKgel G3000SW

7.5 × 300 10 250 1980 3845 3

TSKgel G3000SWxl

7.8 × 300 5 250 5060 10674 4

Shodex KW-804

8.0 × 300 7 250 4952 8859 2

Protein-Pak 300SW

7.5 × 300 10 250 2078 4271 3

BioSuite 250

7.8 × 300 5 250 5149 9403 3

The drug aprotinin (Trasylol, previously Bayer and now Nordic Grouppharmaceuticals), is the small protein bovine pancreatic trypsin inhibitor,or BPTI, which inhibits trypsin and related proteolytic enzymes. Under thetrade name Trasylol, aprotinin was used as a medication administeredby injection to reduce bleeding during complex surgery, such as heart and liversurgery. Its main effect is the slowing down of fibrinolysis, the process thatleads to the breakdown of blood clots. The aim in its use was to decrease theneed for blood transfusions during surgery, as well as end-organ damage dueto hypotension (low blood pressure) as a result of marked blood loss. The drugwas temporarily withdrawn worldwide in 2007 after studies suggested that itsuse increased the risk of complications or death

Schematic representation of some of the key steps in non-native aggregation

Plate heights (HETP) vs. linear velocity (u0) plotsof Panitumumab (A), chicken ovalbumin (B) and β-lactoglobulin (C). Columns: Acquity UPLC BEH200 SEC 1.7 μm, 150 mm × 4.6 mm (operated at 30, and60 °C), YMC-Pack-Diol-200 5 μm, 300 mm × 6 mm andPhenomenex Yarra SEC-3000 3 μm, 300 mm × 4.6 mm (operated at 30 and50 °C). Mobile phase: 20 mM disodiumhydrogen-phosphatebuffer of pH = 6.8.

Journal of Pharmaceutical and Biomedical Analysis 78–79 (2013) 141– 149

Effect of pressure (A) and temperature (B)on the observed aggregates. Column:Acquity UPLC BEH200 SEC 1.7 μm,150 mm × 4.6 mm. Mobile phase: 20 mMdisodium hydrogen-phosphate buffer ofpH = 6.8. Flow rate: 200 μl/min, detection:FL (Ex: 280 nm, Em: 360 nm). The columnpressure (head pressure) was varied byadding restrictor capillaries to the columnoutlet (131, 271, 406 and 465 bar weregenerated, including the column pressure)on (A). Sample: heat stressedpanitumumab.

Representative chromatograms on the effect of column temperature (A) and pressure (B) on the observed amount of antibody aggregates

Representative chromatograms on BEH 1.7 μm column (A) and on YMC diol 5 μm column (B) obtained by injecting the same sample (native panitumumab). Mobile phase: 20 mM disodiumhydrogen-phosphate buffer of pH = 6.8. Flow rate: 500 μl/min, detection: FL (Ex: 280 nm, Em: 360 nm), mobile phase temperature: 30 °C. The generated pressure was 274 bar (A) and 73 bar (B).

Fast separation of the aggregate and native form of β-lactoglobulin (A) and of chicken eggovalbumin (B). Column: Acquity UPLC BEH200 SEC 1.7 μm, 150 mm × 4.6 mm. Mobile phase:20 mM disodium hydrogen-phosphate buffer of pH = 6.8. For β-lactoglobulin: flow rate:700 μl/min, mobile phase temperature: 45 °C and for egg ovalbumin: flow rate: 850 μl/min,mobile phase temperature: 60 °C. Detection: FL (Ex: 280 nm, Em: 360 nm) for both cases. Peaks:1, 2 and 3: high molecular weight species.

C. Wong et al. / J. Chromatogr. A 1270 (2012) 153– 161

SE-HPLC chromatogram profile at 280 nm showing fronting shoulder on monomer of a drug product at time zero (red) and 40 °C 2 months stability sample (black). Column TSKgel BioAsisstG3SWXL

Representative chromatograms of formulated bulk separation using 0.25 M NaCl (black), 0.5 MNaCl (blue), 0.75 M NaCl (green), 1.0 M NaCl (cyan), 1.25 M NaCl (magenta), 1.5 M NaCl(purple), and 1.7 M NaCl (red) sodium chloride in 20 mM sodium phosphate, pH 7.0 mobilephase, and Waters Acquity BHE200 4.6 mm × 300 mm column shown at 280 nm. (Forinterpretation of the references to color in this figure legend, the reader is referred to the webversion of the article.)

Representative chromatogram of (A) formulated bulk material (blue) and purified monomer fraction (black), and (B) 40 °C, 9 months stability sample (blue) and purified pre-peak fraction (black) using the final mixed mode UPLC method at 280 nm.

Mobile phase containing20 mM sodium phosphate atpH 7.0 and a flow rate of0.15 mL/min showed the bestseparation for the mixed modeUPLC method. The pre-peakand the monomer peak werefractionated using the finalmixed mode UPLC method forcharacterization

J. Sep. Sci. 2013, 36, 2718–2727

Chromatograms of PS standard (Mp ∼ 11 600 g/mol, -D- ∼ 1.03) obtained on XBridge(TM) C18 and ACQUITY R C18 columns (4.6 × 150 mm) packed with different size particles: 10, 5, 3.5, and1.7 m. Mobile phase, THF; flow rate, 1 mL/min; detection, UV at 254 nm.

Stacked chromatograms of SEC separation of two proprietary polymers on BEH 45 unbondedand BEH 45 TMS columns, 4.6 × 150mmin THF at 1 mL/min using UV detection at 254 nm. (A) Polymer A on BEH 45 unbonded; (B) polymer B on BEH 45 unbonded; (C) polymer A on BEH 45 TMS; (D) polymer B on BEH 45 TMS

Chromatograms of PSstandards on (A) BEH 200diol, 4.6 × 150 mm,detection, UV at 254 nm;(B) same as (A), but withELS detection. Fivereplicate injections areshown, demonstratingrepeatability; (C) 5 mPLgel MiniMix D (4.6 ×250 mm); detection, UVat 254 nm. In all cases,mobile phase was THFand flow rate was 1mL/min.

Chromatograms of (A) fourteencomponent mixture of PS standards obtained on two 4.6 × 150mm columns connected in series. First column, 1.7 m BEH diol 200 A° ; second column, 1.7 mBEH diol 450 A° . Mobile phase, THF; flow rate, 1 mL/min; detection, ELS, (B) twelve-componentPS standards obtained on three PL gel SEC columns (7.5 × 300 mm each) packed with 5 mPsdivinylbenzene particles with pore sizes labeled as 10E2, 10E3, and 10E4 A° . Mobile phase,THF; flow rate, 1 mL/min; detection, RI.

Chromatograms of PMMA standards and corresponding calibration curve (fifth-order fit). Mobile phase, THF; flow rate, 0.4 mL/min; column, 4.6 × 150 mm, packed with 1.7 mdiol-bonded particles with amean pore size of 200 A° ; detection, ELS.

Efeito do contra ion e de sua concentração na retenção de β-

bloqueadores.

FLIEGER, J. The effect of chaotropic mobile phase additives on the separation of selected alkaloids in reversed-phase high-performance liquid chromatography. Journal of Chromatography A, v. 1113, n. 1, p. 37-44, 2006.

Chromatograms of a mixtures of alkaloids(A—caffenine, B—laudanozine, C—colchicine, D—boldine, E—strychnine, F—cinchonine, G—quinine) with different organicanions in the mobilephase.

Effect of anionic additive type on the retention of investigated alkaloids. (*) For emetine andberberine the strongest retention was observed when hexafluorophosphate salt was added tothe mobile phase. Their retention factors were higher than 25.

The effect of different anionic additives on retention, peak symmetry and efficiency of narcotine.

Jones, Alan, Rosario LoBrutto, and Yuri Kazakevich. "Effect of the counter-anion type andconcentration on the liquid chromatography retention of β-blockers." Journal ofChromatography A 964.1 (2002): 179-187.

Dependence of the retention factors for labetolol, acebutolol, and nadolol versus theconcentration of perchlorate counter-anion in the mobile phase. Chromatographicconditions: column: Zorbax Eclipse XDB-C18 (150×4.6 mm), mobile phase: aqueousadjusted with perchloric acid and/or sodium perchlorate (pH 3.0)–acetonitrile (70:30), flow-rate: 1 ml/min, detection: UV at 225 nm.

Dependence of the retention factors for metoprolol, pindolol, and nadolol versus theconcentration of perchlorate counter-anion in the mobile phase. Chromatographicconditions: column: Zorbax Eclipse XDB-C18 (150×4.6 mm), mobile phase: aqueousadjusted with perchloric acid and/or sodium perchlorate (pH 3.0)–acetonitrile (70:30), flow-rate: 1 ml/min, detection: UV at 225 nm.

Plot of the acebutolol retention factors versus counter-anion concentration in the mobile phasefor different counter-anions used. Chromatographic conditions: column: Zorbax Eclipse XDB-C18 (150×4.6 mm), mobile phase: aqueous (pH 3.0)–acetonitrile (70:30), flow-rate: 1 ml/min, detection: UV at 225 nm.

Plot of the acebutolol retention factors versus counter-anion concentration in the mobile phasefor different counter-anions used. Chromatographic conditions: column: Zorbax Eclipse XDB-C18 (150×4.6 mm), mobile phase: aqueous (pH 3.0)–acetonitrile (70:30), flow-rate: 1 ml/min, detection: UV at 225 nm.

Chromatograms of a mixture of β-blockers and o-chloroaniline analyzed at constant pH and increasing perchlorate concentration.. Chromatographic conditions: column: Zorbax Eclipse XDB-C18 (150×4.6 mm), mobile phase: aqueous (pH 3.0)–acetonitrile (70:30), flow-rate: 1 ml/min, detection: UV at 225 nm.

FLIEGER, J. Effect of mobile phase composition on the retention of selected alkaloids in reversed-phase liquid chromatography with chaotropicsalts. Journal of chromatography A, v. 1175, n. 2, p. 207-216, 2007

Experimental retention factors obtained for investigated alkaloids vs. trifluoroacetate andhexafluorophosphate concentration in mobile phase: 30% ACN/10 mM phosphate buffer (dashed lines) and 30% ACN/30 mM phosphate buffer pH = 2.7 (continuous lines).

Graphic comparison of thedesolvation parametersobtained usinghexafluorophosphate asthe counter-anion for twoeluent systems: 25%THF/30 mM phosphatebuffer (THF) and 30%ACN/30 mM phosphatebuffer (ACN).

Chromatograms of mixtures of alkaloids obtained by the use of different mobile phases: (A) 35% ACN/10 mM phosphate buffer (pH 2.7) + 30 mM NaPF6, (B) 40% MeOH/10 mM phosphatebuffer (pH 2.7) + 30 mM NaPF6, (C) 25% THF/10 mM phosphate buffer (pH 2.7) + 30 mM NaPF6.

PAN, Li et al. Influence of inorganic mobile phase additives on the retention, efficiency and peak symmetry of protonated basic compounds in reversed-phase liquid chromatography. Journal of Chromatography A, v. 1049, n. 1, p. 63-73, 2004.

Effect of analyteload on: (A) N(h/2) and (B) tailing factor. Chromatographicconditions: 0.1% (v/v) H3PO4:acetonitrileeluent; benzylamine (5% acetonitrile), toluene (50% acetonitrile), Labetalol and 4-nitrophenol (25% acetonitrile), flowrate: 1.0 mL/min; temperature: 25 °C; analyte load: 0.5–50 μg.

Chromatographic overlays of Labetalol analyzed at different analyte concentrations usingincreasing mobile phase concentration of perchlorate anion. Chromatographic conditions:analyte load: 3.3, 6.5, 31.2 μg, (a) 75%:0.1% (v/v) H3PO4:25% acetonitrile; (b) 75%:0.05% (v/v)HClO4:25% acetonitrile; (c) 75%:0.3% (v/v) HClO4:25% acetonitrile; (d) 75%:0.4% (v/v)HClO4:25% acetonitrile; (e) 75%:0.5% (v/v) HClO4:25% acetonitrile.

Chromatographic overlays of Dorzolamide HCl analyzed at different analyte concentrations usingincreasing mobile phase concentration of perchlorate anion. Chromatographic conditions:Analyte load: 1.4, 5.2, 9.2, 48 μg, (a) 90%:0.1% (v/v) H3PO4:10% acetonitrile; (b) 90%:0.05% (v/v)HClO4:10% acetonitrile; (c) 90%:0.3% (v/v) HClO4:10% acetonitrile; (d) 90%:0.4% (v/v)HClO4:10% acetonitrile; (e) 90%:0.5% (v/v) HClO4:10% acetonitrile.

Effect of counteranion type and concentration on analyte retention, peak efficiency, N(h/2), andtailing factor. Chromatographic conditions: Mobile phase: 75% aqueous:25% acetonitrile.Effective counteranion concentration for each mobile phase indicated in figure legend, flow rate:1.0 mL/min; temperature: 25 °C; analyte load: 0.5 μg; wavelength: 225 nm.

FLIEGER, J. Application of perfluorinated acids as ion-pairing reagents for reversed-phase chromatography and retention-hydrophobicityrelationships studies of selected β-blockers. Journal of Chromatography A, v. 1217, n. 4, p. 540-549, 2010.

Effect of ion-pairing reagentconcentration inmethanol/water mobilephase (acetic acid, AA;trifluoroacetic acid, TFAA;pentafluoropropionic acid,PFPA; heptafluorobutyricacid, HFBA) on retentioncoefficient of investigatedβ-blockers.

Chromatograms of mixtures of β-blockers obtained by the use of different mobile phases. The peaks order: atenolol, pindolol, nadolol, metoprolol, acebutolol.

SIR mass chromatograms of a mixture of oligolysine (dp = 2–8) at different percentages of ACN in the mobile phase when heptafluorobutyric acid [HFBA] is 9.2 mM. Column Waters XBridge Shield RP18 column (50 mm × 4.6 mm i.d.; pore size 135 Å, particle size 3.5 μm) thermostated at 35 °C. The number on the top of each peak represents dp. Peaks corresponding to dp = 7 and 8 are shown as an inset for 23% ACN.

XIE, Wenchun; TERAOKA, Iwao; GROSS,

Richard A. Reversed phase ion-pairing

chromatography of an oligolysine mixture in

different mobile phases: effort of searching

critical chromatography conditions. Journal

of Chromatography A, v. 1304, p. 127-132,

2013.

Effect of the HFBA concentration on the retention of oligolyisne. The number of lysine residues is indicated adjacent to each curve. (a) Results for all concentrations of HFBA. (b) Results at low concentrations. The y axis is in a log scale in (a) and in a linear scale in (b).

XIE, Wenchun et al. Cooperative effect in ionpairing of oligolysine with heptafluorobutyricacid in reversed-phasechromatography. Journal of ChromatographyA, v. 1218, n. 43, p. 7765-7770, 2011.

LONG, Zhen et al. Strong cationexchange column allow for symmetrical peak shape and increased sample loading in the separation of basic compounds. Journal of Chromatography A, v. 1256, p. 67-71, 2012.

Chromatograms of basic compounds separated on the Sunfire C18 column (A), XBridgeC18 column (B) and the XChargeSCX column (C); Loading amounts on columns were 0.09035 mg, 0.9035 mg, and 3.614 mg from (a) to (c). Peaks: 1 = propranolol, 2 = berberine, 3 = amitriptyline.The mobile phases used for the separation of basic compounds on XCharge SCX column were A: acetonitrile, B: 100 mmol/L NaH2PO4 (pH = 2.83) and C: water. The flow rate was 1.0 mL/min and peaks were recorded at 260 nm. Mobile phase composition on the XCharge SCX column was 50% A, 30% B. The optimized mobile phases on Sunfire C18 column were A: 0.1% FA in ACN (v/v) and B: 0.1% FA in water (v/v). Mobile phase composition on Sunfire C18 column started at 10% A and shifted to 35% A over 30 min. Mobile phases for the analysis of basic compounds on XBridge C18 column were A: acetonitrile, B: 100 mmol/L NH4HCO3 (pH adjusted to 10.12 with ammonia solution) and C: water. Mobile phase composition on XBridge C18 column started at 20% A, 10% B, shifted to 30% A, 10% B from 0 to 10 min, and finally shifted to 60% A, 10% B from 10 to 40 min.

Detectores

Universidade Federal do Rio Grande do NorteCentro de Ciências Exatas e da Terra

Instituto de QuímicaMatéria de ensino: Química Analítica

DETECTORESClassificação

UNIVERSAIS:Geram sinal para qualquer

substância eluida.

SELETIVOS:Detectam apenas substâncias

com determinada propriedadefísico-química.

ESPECÍFICOS:Detectam substâncias que

possuam determinado elementoou grupo funcional em suas

estruturas

DETECTORESParâmetros Básicos de Desempenho

SENSIBILIDADE Relação entre o incremento de área do pico e o incremento de massa do analito

MASSA

ÁR

EA

Fator de Resposta, S: inclinação da reta

Área do pico x Massa do analito

o mesmo incremento de massa causa um maior

incremento de áreaSensibilidadeS

Na ausência de erros determinados:

A = área do pico cromatográfico

m = massa do analito

FAIXA LINEAR DINÂMICA Intervalo de massas dentro do qual a resposta do detector é linear

MASSA

ÁR

EA

A partir de certo ponto o sinal não

aumenta mais linearmente

O fim da zona de linearidade pode ser detectado quando a razão (Área / Massa)

diverge em mais de 5 % da inclinação da reta na

região linear:

MASSA

ÁR

EA /

MA

SSA

0,95 S

1,05 S

Detector de Ultra violeta (ultraviolet detector, UV) ou espectroquímico

Extrato de folhas da M. aquifolium depois de um processo de hidrolise, detecção a 270 nm

Chromatography Research Inte national Volume 2012 (2012), Article ID 691509, 7 pages

http://dx.doi.org/10.1155/2012/691509

Uma solução 0,1 mol/L de umasubstância de coloração intensa éanalisada por LC-UV, mas nenhumpico é observado. Por que?

Detector de índice de refração (refractiveindex detector, RI)

(A) Indice de refração quando não há analito e (B) quando há analito.

G. Iriarteet al.,J. Sep. Sci.,29, 2265 (2006)

Detector Fluorescência.

Fontes de ionização para o LC-MS

• Ionização por eletronebulização – ESI(Electrospray)

– Modo ESI

– Modo Ionspray (ISI)

• Ionização Química a Pressão Atmosférica – APCI

• Fotoionização a Pressão Atmosférica – APPI

Espectrometria de massas (mass spectrometry, MSDiagramas de blocos de um MS

ALTO VÁCUO

Interface / Fonte de Íons

Analisadorde Massas

Detector

ALTO VÁCUO

Interface / Fonte de Íons

Analisadorde Massas

Detector

Modo de Ionização à Pressão Atmosférica (API)

Modo de Ionização no Vácuo

Introdução dos analitos

Introdução dos analitos

Exemplos: EI, CI, PI, MALDI, TSI, FAB, SIMS, FI/FD

Exemplos: ESI, APCI, APPI, AP-MALDI, DESI, DART, EASI

Escolhendo o modo de ionização e a polaridade

ESI: solutos iônicos e polares (PM 100-150x103 dalton).

Moléculas maiores adquirem mais que uma carga.

APCI: solutos de polaridade média e não-polares

(PM 100-2000 dalton).

APPI: mesmos solutos que APCI mas resposta melhor para moléculas de alta apolaridade.

Electrospray – ESI

Vazões: 100 nL/min a 5 µL/min

Como você acha que a percentagem de modificador orgânico na fase móvel?

ESI pode ser usado com que modos da cromatografia líquida NP, RP, IEX, SE e HILIC?

HILIC: A Critical Evaluationhttp://www.chromatographyonline.com/lcgc/article/articleDetail.jsp?id=835539&pageID=1

Ionização Química a Pressão Atmosférica

(APCI)

Saída HPLC

Gás de nebulizaçãoNebulizador (sprayer)

Vaporizador (aquecedor)

Gás de secagem

Capilar

Lâmpada UV

Fotoionização a Pressão Atmosférica (APPI)

• Pode ser usado um dopante

Ex. Tolueno

http://www.shimadzu.com/an/lcms/lcmsittof/ittof-8.html

Análise de benzo[a]pireno;100 picomoles; modo positivo; injeção em fluxo.

Baixa abundância do íon [M+H]+ em m/z 253.

Íon [M+H]+ mais abundante

Presença de M+ em m/z 252

Isótopo C13 em m/z 254

Abundância 5 x maior que APCI e 20 x maior que ESI.

Agilent Technologies, www.agilent.com/chem, 2001.

Ésteres de testosterona: baixo PM e polaridade média

Que modo de ionização vc escolheria?

ESI ou APCI?

Ésteres de testosterona: baixo PM e polaridade média

1 propionato de testosterona

2 fenilpropionato

3 caproato

4 decanoato

(b) Pico 2 em APCI

(c) Pico 2 em ESI

Sandra, P. et al., LC-GC Europe, 2001.

Modos de ionização para MS(/MS)

Mas

sa M

ole

cula

r

Apolar

Polaridade

Muito Polar

Fonte: Agilent Technologies, www.agilent.com/chem, 2001.

(GC) EI(GC) CI

Analisadores para MS

• Setor Magnético e Setor Eletrostático

• Quadrupolo

• Ion Trap

• Tempo de Voo

• FT-ICR

• (FT) Orbitrap

Focalização Dupla

• Vantagens– Alta resolução e exatidão

– Excelente estabilidade = resultados quatitativos

– Permite MS/MS

• Desvantagens– Caro -- Difícil de usar

– Velocidade de varredura limitada (histerese)

– Detectabilidade é dependente da velocidade

Tempo de Voo - TOF

Quadrupolo

- Espectro dos produtos ou

“íons filhos”

Modos de operação

MS1

Estático

Massa do precursor (pai)

Câmara de colisão

Fragmentação CID

Modo RF (passam todas as massas)

MS2

Scan

Espectro dos produtos (ions

filhos)

MRM (frente) x SIM (trás)

Mercaptobenzotiazol

SIM: m/z 166

MRM: m/z 166 → 134

Mercaptobenzoxazol

SIM: m/z 150

MRM: m/z 150 → 58

Trends Anal. Chem., 2001, 20, 533-542.

QqQ – Aplicação

Tempo de Retenção, minutos

Salinomicina

Narasina

Nigericina

Lasalocida

Monensina

Cromatograma obtido a partir da análise de matriz fortificada com LAS (20 µg kg-1), MON (10 µg kg-1), SAL (100 µg kg-1) e NAR (15 µg kg-1) na concentração de seus respectivos LMRs e NIG (50 µg kg-1).