Cromatografia líquida moderna

O que é cromatografia líquida?

•http://chemkeys.com/br/2000/07/18/cromatografia-a-gas-curso-em-diapositivos/
CROMATOGRAFIA
Histórico
M. TSWEET (1903): Separação de misturas de
pigmentos vegetais em colunas recheadas com
adsorventes sólidos e solventes variados.
éter de
petróleo
CaCO3
mistura de
pigmentos
pigmentos
separados
Cromatografia =
kroma [cor] + graph [escrever]
(grego)
http://chemkeys.com/br/2000/07/18/cromatografia-a-gas-curso-em-diapositivos/

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

CROMATOGRAFIA
Princípio Básico
Separação de misturas por interação diferencial dos seus
componentes entre uma FASE ESTACIONÁRIA (líquido ou
sólido) e uma FASE MÓVEL (líquido ou gás).

CROMATOGRAFIA
Modalidades 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)

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

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

Parâmetros cromatográficos que você deve conhecer,
quando trabalha com cromatografia.

Cromatograma com as medidas relacionadas à determinação de parâmetros cromatográficos
C.H. Collins, G.L. Braga, P.S. Bonato,
Fundamentos de Cromatografia, 4ª
edição .,:Editora da Unicamp, Campinas,
2006.
R
M
t t
R M
M
t
t
t
k
'



2
1
2
  
1
'
R
t
'
R
t
k
k

 


t t
R R
2 1 2 1,177
 
s w w


R R
2 1


  


 




1 2
1 2
h h
b b
t t
w w
R

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-Interscience
Hoboken, 2005.

L
N
H 
2 2




R
R
t
t
545 , 5 16  

 

  

 


h
b
w
w
N

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

C

B
H  A 

L
M t
 
http://www.chromedia.org/chromedia?waxtrapp=yqegzCsHqnOxmOlIEcCbC&subNav=wnjedDs
HqnOxmOlIEcCbCmF

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.

푑푝 2휇
퐷푀
푣 =
a)
+ 푐푣
b)
퐻 = 퐴푑푝 +
퐵퐷푀
휇
+ 퐶
휇푑푝
퐷푚
ℎ =
퐻
푑푝
ℎ = 푎 +
푏
푣
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

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

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

Quais são os principais modos de cromatografia?

http://www.chromedia.org/chromedia?
waxtrapp=yqegzCsHqnOxmOlIEcCbC&su
bNav=yarwnEsHqnOxmOlIEcCzBYT
Different mechanisms of retardation in
liquid chromatography (J.F. Rubinson, K.A.
Rubinson, Contemporary Chemical
Analysis, Prentice Hall, Upper Saddle
River, 1998).

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 phthalic
anhydride with benzene in the presence of AlCl3, as catalyst. Peaks: 1, benzene; 2,
anthraquinone; 3, phthalic anhydride; 4, maleic anhydride; 5, unknown. Chromatographic
conditions: 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.

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/water
eluent.

%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 de
retenção (k) e seletividade (α) do
nitrobenzeno (NB) e do
propilparabeno (PP) em função
da percentagem e do tipo de
modificador orgânico. Coluna:
Symmetry C18, 3 μm, 75 × 4,6
mm, 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-phase
component. Chromatograms showing the elution order of all eight
congeners as a function of organic modifier with 0.1% formic acid as
the 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 reference
51, 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 0
4 55 45
40 0 100
45 0 100
48 100 0
A. Stafiej et al. / J. Biochem. Biophys. Methods 69 (2006)
15–24

Theoretical retention versus pH profiles for acidic and a zwitterionic component. Chromatographic
conditions: 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

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

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

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

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

H. Engelhardt, R. Grüner, M. Schererv
Chromatographia, 53 (2001), pp.
S154–S161
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.

O que acontece quando usamos partículas com
diâmetro reduzido?

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).

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).

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 pharmaceutical
formulation “Rapidocain” in
isocratic mode. Experimental
conditions: mobile phase
containing 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 μm
particles 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
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 with
different lengths. Conditions: 50,
100, and 150 mm BEH Shield C-18
columns packed with 1.7, 3.5, and
5.0 μm particles 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
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.

Loss in efficiency at various retention factors for columns with different
lengths. 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 μm
column with A. a
conventional HPLC system
without modification, and B.
after optimization.
https://phenomenex.blob.c
ore.windows.net/document
s/ad6882fd-5ad1-478a-ab0a-
5ad746734529.pdf (06.02.2
013).

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

Jul 1, 2012
By: Fabrice Gritti, Georges Guiochon
LCGC 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 trifluroacetic
acid 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 μm
column 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 phosphate
and 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 (generated
by restrictor tubes at column
outlet) on the retention of
small analytes. Column: Acquity
BEHC18 (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) and
testosterone (6).
S. Fekete et al. / J.
Chromatogr. A 1270 (2012)
127– 138

Effect of pressure (generated by
restrictor tubes at column outlet) on
the retention of related peptides
(1–1.3 kDa). Column: Acquity
BEH300 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, injected
volume: 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 the
combination of pressure and
mobile phase velocitiy (B) on
the retention (relative
change in k) of different size
analytes (1.1 kDa peptide,
5.7 kDa insulin, 12.3 kDa
cytochrome C and 17 kDa
myoglobin). Column: Acquity
BEH300 C18
(50 mm × 2.1 mm), mobile
phase A: water (0.1% TFA),
mobile phase B: acetonitrile
(0.1% TFA). Detection:
fluorescence ex.: 280 nm,
em.: 360 nm. Investigated
pressure range: 100–
1100 bar.

Effect of very high
pressure (generated by
restrictor tubes at
column outlet) on the
retention (A) and peak
shape (A and B) of
cytochrome 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: fluorescence
ex.: 280 nm, em.:
360 nm.

Effect of pressure on
selectivity of separation of a
diverse mixture of
compounds Column: Ace
Excel 5 C18, 5 cm × 0.21 cm
I.D., 5 μm, mobile phase 25%
ACN in 0.025 M phosphate
buffer 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 the
Acquity system.
M.M. Fallas et al. / J.
Chromatogr. A 1297 (2013)
37– 45

Wide-pore fused-core particles for protein
separations. 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: carbonic
anhydrase (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 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: carbonic
anhydrase (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; mobile
phase B: acetonitrile with 0.1% trifluoroacetic acid; flow rate: 0.45 mL/min; instrument: Agilent
1200 SL; injection volume, 2 μL; detection: 215 nm; temperatures as indicated; solutes, proteins
as 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; flow
rate: 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. Peak
widths 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 starting
reaction 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 controlling
the mesopore size: 0.01 M. The skeleton size and through-pore size are indicated by black and white
arrows, respectively, in (a). Journal of Chromatography A, 1191 (2008) 231–252

Experimental Van Deemter
plots of 2.6 μm shell-type
(Kinetex), 2.7 μm shell-type
(Ascentis Express), sub-
2 μm totally porous
particles and a monolith
column (peak widths were
corrected for the extra-column
broadening).
Mobile phase: (A) 48%
acetonitrile–52% water for
estradiol and (B) 95%
acetonitrile–5% water for
ivermectin, 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 thiols
Analyst,2013, 138, 3802-
3808.

Effect of the type of alcohols on the retention of hydrazines on a ZIC HILIC column. For each
separation, 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. For
each 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 splitter
and 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) Zorbax
NH2, (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 of
0.5 mL/min., λ = 249 nm,
injected volume = 1 μL.
Condition: pH 3
ammonium formate
50 mmol/L, pH 4
ammonium formate
50 mmol/L, pH 5
ammonium acetate
50 mmol/L, pH 6
ammonium acetate
50 mmol/L, gradient
profile: 95% ACN for
1 min, then 95–65% ACN
in 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 on
selectivity: ACN, ACN/IPA 80:20
v/v, and ACN/MeOH 80:20 v/v.
Column: silica bare, flow rate of
0.5 mL/min, λ = 249 nm,
injected volume = 1 μL.
Condition: ammonium formate
(50 mmol/L, pH 4), gradient
profile: 5% buffer for 1 min,
then 5–35% buffer in 3 min
with 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 a
mixture of hypoxanthine (1: 80 μg/mL), cytosine (2: 10 μg/mL), nicotinic acid (3: 30 μg/mL) and
procainamide (4: 30 μg/mL) dissolved in pure ACN. Conditions: mobile phase: ammonium
formate (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 of
500 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); the
star (*) 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), flow
rate 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.

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 the
gas constant: T is absolute temperature, and k is the partition coefficient. For most
chromatographic modes of separation, the enthalpy of adsorption is the dominant contributor
to the overall change in free energy. SEC is unique in that partitioning is driven entirely by
entropic processes as there ideally is no adsorption, ΔH = 0. Thus the previous equation
becomes: 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 impact
retention to a small degree by altering the conformation of the proteins, as well as by affecting
mobile phase viscosity and analyte diffusivity.

The thermodynamic SEC retention factor is the fraction of intraparticle pore
volume that is accessible to the analyte: where V R , V 0, and V i are the respective
retention 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 excluded
from the pores of the stationary phase, to a value of 1 where the analyte fully
accesses 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). Reproduced
with permission from Waters Corporation, Milford, MA

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 monoclonal
antibody (blue) on two columns varying in particle size. The 4.6 × 150 mm columns were packed
with either 1.7 micron (solid line) or 2.6 micron particles (dashed line). Pore size of stationary
phase 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
Dimension
s (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 Group
pharmaceuticals), is the small protein bovine pancreatic trypsin inhibitor,
or BPTI, which inhibits trypsin and related proteolytic enzymes. Under the
trade name Trasylol, aprotinin was used as a medication administered
by injection to reduce bleeding during complex surgery, such as heart and liver
surgery. Its main effect is the slowing down of fibrinolysis, the process that
leads to the breakdown of blood clots. The aim in its use was to decrease the
need for blood transfusions during surgery, as well as end-organ damage due
to hypotension (low blood pressure) as a result of marked blood loss. The drug
was temporarily withdrawn worldwide in 2007 after studies suggested that its
use 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) plots
of 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, and
60 °C), YMC-Pack-Diol-
200 5 μm,
300 mm × 6 mm and
Phenomenex Yarra SEC-
3000 3 μm,
300 mm × 4.6 mm
(operated at 30 and
50 °C). Mobile phase:
20 mM disodium
hydrogen-phosphate
buffer 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 mM
disodium hydrogen-phosphate buffer of
pH = 6.8. Flow rate: 200 μl/min, detection:
FL (Ex: 280 nm, Em: 360 nm). The column
pressure (head pressure) was varied by
adding restrictor capillaries to the column
outlet (131, 271, 406 and 465 bar were
generated, including the column pressure)
on (A). Sample: heat stressed
panitumumab.

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 disodium
hydrogen-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 egg
ovalbumin (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 BioAsisst
G3SWXL

Representative chromatograms of formulated bulk separation using 0.25 M NaCl (black), 0.5 M
NaCl (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 mobile
phase, and Waters Acquity BHE200 4.6 mm × 300 mm column shown at 280 nm. (For
interpretation of the references to color in this figure legend, the reader is referred to the web
version 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 containing
20 mM sodium phosphate at
pH 7.0 and a flow rate of
0.15 mL/min showed the best
separation for the mixed mode
UPLC method. The pre-peak
and the monomer peak were
fractionated using the final
mixed mode UPLC method for
characterization

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, and
1.7 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 unbonded
and 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 PS
standards on (A) BEH 200
diol, 4.6 × 150 mm,
detection, UV at 254 nm;
(B) same as (A), but with
ELS detection. Five
replicate injections are
shown, demonstrating
repeatability; (C) 5 m
PLgel MiniMix D (4.6 ×
250 mm); detection, UV
at 254 nm. In all cases,
mobile phase was THF
and flow rate was 1
mL/min.

Chromatograms of (A) fourteencomponent mixture of PS standards obtained on two 4.6 × 150
mm columns connected in series. First column, 1.7 m BEH diol 200 A° ; second column, 1.7 m
BEH diol 450 A° . Mobile phase, THF; flow rate, 1 mL/min; detection, ELS, (B) twelve-component
PS standards obtained on three PL gel SEC columns (7.5 × 300 mm each) packed with 5 m
Psdivinylbenzene 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 organic
anions in the mobile
phase.

Effect of anionic additive type on the retention of investigated alkaloids. (*) For emetine and
berberine the strongest retention was observed when hexafluorophosphate salt was added to
the 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 and
concentration on the liquid chromatography retention of β-blockers." Journal of
Chromatography A 964.1 (2002): 179-187.

Dependence of the retention factors for labetolol, acebutolol, and nadolol versus the
concentration of perchlorate counter-anion in the mobile phase. Chromatographic
conditions: column: Zorbax Eclipse XDB-C18 (150×4.6 mm), mobile phase: aqueous
adjusted 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 the
concentration of perchlorate counter-anion in the mobile phase. Chromatographic
conditions: column: Zorbax Eclipse XDB-C18 (150×4.6 mm), mobile phase: aqueous
adjusted 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 phase
for 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 chaotropic
salts. Journal of
chromatography A,
v. 1175, n. 2, p. 207-
216, 2007

Experimental retention factors obtained for investigated alkaloids vs. trifluoroacetate and
hexafluorophosphate 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 the
desolvation parameters
obtained using
hexafluorophosphate as
the counter-anion for two
eluent systems: 25%
THF/30 mM phosphate
buffer (THF) and 30%
ACN/30 mM phosphate
buffer (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 phosphate
buffer (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 analyte
load on:
(A) N(h/2) and (B)
tailing factor.
Chromatographic
conditions: 0.1%
(v/v)
H3PO4:acetonitrile
eluent;
benzylamine (5%
acetonitrile),
toluene (50%
acetonitrile),
Labetalol and 4-
nitrophenol (25%
acetonitrile), flow
rate: 1.0 mL/min;
temperature: 25 °C;
analyte load: 0.5–
50 μg.

Chromatographic overlays of Labetalol analyzed at different analyte concentrations using
increasing 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 using
increasing 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), and
tailing 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-hydrophobicity
relationships studies of
selected β-blockers. Journal
of Chromatography A, v.
1217, n. 4, p. 540-549, 2010.
Effect of ion-pairing reagent
concentration in
methanol/water mobile
phase (acetic acid, AA;
trifluoroacetic acid, TFAA;
pentafluoropropionic acid,
PFPA; heptafluorobutyric
acid, HFBA) on retention
coefficient of investigated
β-blockers.

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

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.
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 et al. Cooperative effect in ion
pairing of oligolysine with heptafluorobutyric
acid in reversed-phase
chromatography. Journal of Chromatography
A, v. 1218, n. 43, p. 7765-7770, 2011.
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).

LONG, Zhen et al. Strong cation
exchange 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), XBridge
C18 column (B) and the XCharge
SCX 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.

Universidade Federal do Rio Grande do Norte
Centro de Ciências Exatas e da Terra
Instituto de Química
Matéria de ensino: Química Analítica
Detectores

DETECTORES
Classificação
UNIVERSAIS:
Geram sinal para qualquer
substância eluida.
SELETIVOS:
Detectam apenas substâncias
com determinada propriedade
físico-química.
ESPECÍFICOS:
Detectam substâncias que
possuam determinado elemento
ou grupo funcional em suas
estruturas

DETECTORES
Parâmetros Básicos de Desempenho
SENSIBILIDADE Relação entre o incremento de área do pico e o
incremento de massa do analito
MASSA
ÁREA
Fator de Resposta,
S: inclinação da reta
Área do pico x
Massa do analito
o mesmo incremento de
massa causa um maior
incremento de área
S Sensibilidade
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
ÁREA
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
ÁREA / MASSA
1,05 S
0,95 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 uma
substância de coloração intensa é
analisada por LC-UV, mas nenhum
pico é observado. Por que?

Detector de índice de refração (refractive
index detector, RI)
(A) Indice de refração quando não há analito e (B) quando há analito.

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

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, MS
Diagramas de blocos de um MS
Modo de Ionização no Vácuo
Interface /
Fonte de Íons
Interface /
Fonte de Íons
Analisador
de Massas
ALTO VÁCUO
Detector
ALTO VÁCUO
Analisador
de Massas
Detector
Modo de Ionização à Pressão Atmosférica (API)
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 Evaluation
http://www.chromatographyonline.com/lcgc/article/articleDetail.jsp?id=835539&pageID=1

Ionização Química a Pressão Atmosférica
(APCI)

Fotoionização a Pressão Atmosférica (APPI)
Saída HPLC
Gás de nebulização
Nebulizador (sprayer)
Vaporizador (aquecedor)
Gás de secagem
Capilar
Lâmpada UV
• Pode ser
usado um
dopante
Ex. Tolueno

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

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.
Análise de benzo[a]pireno;100 picomoles; modo positivo; injeção em fluxo.
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)
Massa Molecular
(GC) EI
(GC) CI
Apolar
Polaridade
Muito Polar
Fonte: Agilent Technologies, www.agilent.com/chem, 2001.

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

Modos de operação
- Espectro dos produtos ou
“íons filhos”
MS1
Estático
Massa do
precursor (pai)
Câmara de
colisão
Fragmentação CID
Modo RF (passam
todas as massas)
MS2
Scan
Espectro dos
produtos (ions
filhos)

QqQ – Aplicação
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
Tempo de Retenção, minutos
Trends Anal. Chem., 2001, 20, 533-542.

Nigericina
Salinomicina
Narasina
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).

Cromatografia líquida moderna

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