chapter 5: development and optimization of a system for ... 5.pdflc × lc-uv/ms for the separation...

LC × LC-UV/MS for the separation of complex samples

109

Chapter 5: Development and optimization of a system

for comprehensive two-dimensional liquid

chromatography with UV and mass spectrometric

detection for the separation of complex samples by

multi-step gradient elution

Journal of Chromatography A, 1188 (2008) 216–226

Chapter 5

110

Abstract

Comprehensive two-dimensional liquid chromatography (LC × LC) is a powerful tool for

the separation of complex biological samples. This technique offers the advantage of

simplified automation and greater reproducibility in a shorter analysis time than off-line

two-dimensional separation systems. In the present study, an LC × LC system is

developed enabling simultaneous UV and MS detection, and which can be easily

converted to a conventional reversed-phase LC-UV/MS system. In LC × LC, a 60-min

reversed-phase LC separation with a linear solvent gradient in the first dimension is

coupled to a second-dimension separation on a mixed-mode cation-

exchange/reversed-phase column with a modulation time of 60 s. The isocratic

separation in the second-dimension column is optimized by the use of a multi-step

gradient where the organic and the ionic modifier are varied independently. Intraday (n

= 3) and interday (n = 4) variability of the retention times were evaluated with the

complete system and found to be 0.5% and 0.7%, respectively. Good linearity was

observed in calibration curves for three different compounds varying in polarity.


111

Introduction

Comprehensive separation of complex mixtures, e.g., urine samples for metabolomics

or biomarker studies, is a difficult challenge due to the presence of thousands of

components that vary from polar to non-polar and from picomolar to millimolar

concentrations, and that show diverse physico-chemical properties, e.g., acid–base

properties, stability, solubility, detectability. The potential of conventional separation

techniques such as liquid chromatography (LC) or gas chromatography (GC) and

detection approaches like UV–vis or mass spectrometric (MS) detection are limited. In

the last decade, comprehensive multidimensional separation techniques such as GC ×

GC [1,2], LC × GC [3,4], LC × capillary electrophoresis (CE) [5,6], and LC × LC [7–

9,31] have been developed and reported. These multidimensional techniques offer an

enormous separation power and are therefore ideally suited for the analysis of very

complex mixtures. Due to its compatibility with biological matrixes, HPLC is most

widely applied in biological applications. Therefore, the use of comprehensive two-

dimensional liquid chromatography (LC × LC) would be a logical choice for the

separation of complex biological samples where conventional LC does not provide

sufficient separation power. In contrast to multidimensional heart-cutting separation

techniques (LC–LC), where a particular fraction of the first-dimension separation is

transferred and re-separated on the second-dimension column [10], in LC × LC the

entire first dimension is analyzed in the second-dimension separation. Comprehensive

LC × LC offers various advantages over both multidimensional off-line and

conventional separation techniques, especially with respect to enhanced peak capacity,

automation potential, and reproducibility and shorter analysis time. Given the

numerous liquid separation modes, there are numerous possible combinations of first-

and second-dimension separation in LC × LC.

In many of the recent papers on LC × LC, compounds are separated by two

independent separation techniques featuring the same separation mechanism,

especially reversed-phase LC × reversed-phase LC systems (RPLC × RPLC) [11–

15,31]. In these cases, the two-dimensional separation is based on the use of different

organic modifiers and/or RPLC columns with different properties. Nevertheless, due to

the fact that the retention mechanisms in the first and second dimension are largely

the same, components with a low retention factor in the first dimension will still have a

low capacity factor in the second dimension. As a result, the high theoretical peak

capacities can never be attained in such a setup.

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112

Therefore, the use of two different separation mechanisms in the first and second

dimension is generally preferred. A variety of LC × LC separation approaches has been

described, including RPLC × size-exclusion chromatography (SEC) [16–18], normal-

phase (NP) LC × RPLC [7,19], SEC × RPLC [20,21], ion-exchange chromatography

(IEC) × SEC [22], IEC × RPLC [23–25]. IEC × RPLC would be a logical column

combination for the separation of biological samples because of the presence of many

ionic components that can be separated by IEC. Most LC × LC systems based on IEC ×

RPLC are operated with IEC in the first dimension and RPLC in the second dimension.

Perhaps the most widely applied comprehensive LC × LC approach, i.e., the

multidimensional protein identification technology (MudPIT) developed by the group of

Yates [26] and two-column approaches based on this design [27,28], is based on IEC

× RPLC, but in general peptide separation can be considered to be significantly

different from the small-molecule separations as under development in the present

study. Nevertheless, IEC × RPLC would be attractive because RPLC on C18-materials is

the most frequently applied LC mode. RPLC operating conditions are readily compatible

with important LC detectors, UV, fluorescence and especially MS. Due to its selectivity

and sensitivity, and its ability to confirm compound identity or even elucidate unknown

structures, MS detection is nowadays essential in the majority of the LC applications.

From this point of view, the alternative approach of RPLC × IEC, although attractive for

its comprehensive two-dimensionality, would be less favorable, because IEC generally

requires relatively high salt concentrations and is therefore less compatible with MS

detection.

An interesting alternative would be the use of mixed-mode columns featuring both

RPLC and IEC separation mechanisms in the second dimension, as these can eliminate

some of the shortcomings of both RPLC and IEC.

In the present paper, we proposed a novel approach to overcome these limitations by

implementing a mixed-mode RPLC/cation-IEC column in the second dimension in

combination with step-gradient elution. Using a test-set of 32 compounds covering a

very wide range of polarities, we describe method development and optimization

separately for each separation dimension. Optimization of the second-dimension

separation indicated the necessity of the use of a step gradient. The fully automated LC

× LC system has been coupled to a mass spectrometer.


113

Experimental

Chemicals and reagents

Alanine, creatinine, dopamine, caffeine, theophylline, paracetamol,

desmethyldiazepam, midazolam, aspirin, aniline, 2,6-dimethylaniline, ibuprofen,

vanillin, thiobenzamide, nadolol, hydrocortisone, naproxen, diazepam, oxazepam,

sulfamethoxypyridazine, sulfaguanidine, 3,4-hydroxybenzoic acid, sulfamic acid,

imipramine, tryptophan, tyrosine, nitrazepam, carbamazepine, benzothiophen,

metamitron, prometryn, and desethylatrazine were all purchased from Sigma,

Zwijndrecht, The Netherlands. Acetonitrile (ACN), formic acid (FA), acetic acid, and

trifluoroacetic acid (TFA) were purchased from Biosolve, Valkenswaard, The

Netherlands.

The test-set was diluted in 1% (v/v) of ACN in water with 0.1% (v/v) FA at

concentrations of 125 µM for all compounds. The urine sample was collected from a

volunteer; the sample was centrifuged at 13 200 rpm with an eppendorf centrifuge

(VWR International, Amsterdam, The Netherlands) directly after collection and stored

at −20 ◦C.

Instrumentation for LC × LC

A schematic diagram of the setup of the comprehensive LC × LC system is illustrated in

Figure 5.1. The system consists of two parts which can also be used independently as

two separate conventional LC systems.

The first-dimension separation system consisted of a Shimadzu HPLC system (‘s

Hertogenbosch, The Netherlands) comprising of two LC-10AD-VP pumps, a SCL-

10ADvp system controller, a DGU-14A degasser, a SIL-10ADvp autosampler, a CTO-

ACvp column oven. For UV detection, a Kratos absorbance detector spectroflow 757

(Nieuwerkerk ad IJssel, The Netherlands) and/or an Agilent Technologies HP1050

photodiode array detector (DAD) system (Amstelveen, The Netherlands) were used.

The RPLC column was a Luna C18(2) (50 mm × 1 mm I.D.; 3 µm particle size) with a

RP-18 (2 mm × 2 mm) pre-column, purchased from Bester (Amstelveen, The

Netherlands). Pumps, autosampler, column are connected using 0.125 mm I.D.

polyetheretherketone (PEEK) tubing. Post-column, the flow-rate is split in a 1:1 ratio

by means of a T-piece and two 40 cm lengths of 0.064 mm I.D. PEEK tubing. Gradient

elution was performed mixing solvent A (from pump P1) consisting of 1% (v/v) of ACN

in water with 0.1% (v/v) FA and solvent B (from P2) consisting of 90% (v/v) of ACN in

water with 0.1% (v/v) FA at a total flow-rate of 50 µL/min. The final gradient program

Chapter 5

114

was: from 0 to 12 min isocratic at 100% A, from 12 to 50 min a linear gradient 100%

A to 20% A, and from 51 to 60 min isocratic at 100% A.

Figure 5.1: Schematic diagram of the setup of the LC × LC-UV/MS system. P1, P2: first-dimension gradient pumps. P3, P4, P5: second-dimension gradient pumps. AS1, AS2: autosampler. S: a ten-port switching valve with two sample loops of 25 µL (L1, L2). The first-dimension column is a Luna C18(2) (50 mm

× 1.0 mm), the second-dimension column is a primesep A (30 mm × 4.0 mm). Detectors: UV-detector at 220 nm for the first dimension, DAD for the second dimension, and a Q-TOF2 for APCI-MS.

The injection volume was either 5 or 25 µL, depending on the experiment performed.

The column temperature was kept constant at 40 °C. The UV detector was set on

wavelengths of 220 and 254 nm. LC–MS was performed in atmospheric-pressure

chemical ionization (APCI) mode on a Waters/Micromass QTOF2 mass spectrometer

equipped with an APCI interface probe (Waters, Etten-Leur, The Netherlands). When

applied in combination with the first-dimension column, the probe temperature was set

on 300 °C and the ion source temperature on 100 °C, the corona current and the cone

voltage were set at 3.0 µA and 25 V, respectively. The second-dimension separation

system consisted of a Shimadzu HPLC system (‘s Hertogenbosch, The Netherlands)

comprising of a LC-10AD-VPi and two LC-10ADvp pumps, CTO-ACvp column oven, and

an Agilent Technologies HP1050 DAD detector (Amstelveen, The Netherlands). The

mixed-mode column was packed in-house (30 mm × 4 mm I.D.) packed with Primesep


115

A material (5 µm particle size), purchased from Aurora Borealis Control (Schoonebeek,

The Netherlands). In the step gradient at the second-dimension column, three solvents

were applied: Solvent C (from P3) consisting of 1% (v/v) of ACN in water with 0.01%

(v/v) TFA, solvent D (from P4) consisting of 1% (v/v) of ACN in water with 0.5% (v/v)

TFA, and solvent E (from P5) consisting of 10% (v/v) of water in ACN with 0.01% (v/v)

TFA (see below for details). The flow-rate at the second-dimension column was 1.5

mL/min. The column temperature was kept constant at 40 °C. LC–MS was performed

on the same Q-TOF2 mass spectrometer in APCI mode (probe temperature 450 °C

instead of 300 °C).

LC × LC methodology

In the LC × LC setup (see Figure 5.1), the flow-rate from the first-dimension column

was split 1:1, with 25 µL/min to the UV detector and 25 µL/min being transferred to

the second-dimension column. The columns were connected by an electronically

controlled two-position ten-port Rheodyne valve (S in Figure 5.1) which was

pneumatically actuated using compressed air at 7 bar. The valve contained two sample

loops (L1 and L2 in Figure 5.1) of 12.73 cm × 0.50 mm I.D. PEEK tubing with an

internal volume of 25 µL each. The two sample loops were used simultaneously and

alternately: while one loop (loading position) collected a 25-µL fraction of the first-

dimension RPLC column eluate, the other (injection position) released and injected the

previously collected fraction onto the second-dimension Primesep A column. Switching

between the two sample loops was performed every 60 s (duration of the modulation

cycle). This alternating process is continuously applied throughout the entire LC × LC

run.

During LC × LC experiments, chromatographic data acquisition was performed using

Agilent Technologies Chemstation software version 1.04. After acquisition, the data

were exported to a comma-separated value file and converted by an in-house written

two-dimensional (2D) converter program which converts the data into a matrix with

rows corresponding to a 60 s duration and data columns covering all successive

second-dimension chromatograms. Contour representations of the 2D chromatograms

were visualized with Transform version 3.4 which is part of Neosys (CREASO,

Apeldoorn, The Netherlands).

Experiments were performed to optimize the mobile-phase composition of the second-

dimension separation by means of a step gradient, where the ACN and TFA

concentrations were varied independently. In total, 12 different conditions (as detailed

below) were compared with an ACN concentration of 20, 40 or 60% and a TFA

concentration of 0.05, 0.10, 0.15 or 0.20%.

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116

The intraday (n = 3) and interday (n = 4) variability of the retention time of the

complete LC × LC system was determined. In addition, calibration curves were

measured with three components with different polarities: caffeine, metamitron and

hydrocortisone. The achievable detection limits were determined for the same three

compounds. In these experiments, the LC × LC system was operated with the usual

linear gradient in the first-dimension separation and fixed isocratic ACN and TFA

concentrations of 40% and 0.05%, respectively, in the second-dimension separation.

Results and discussion

The development and optimization of a comprehensive two-dimensional LC × LC

system requires a number of steps and considerations, e.g., concerning the choice of

stationary and mobile phases, column dimensions, injection volumes, sample transfer

between the two columns, and optimization of the experimental conditions like mobile-

phase composition, gradients and flow-rates. The final aim of our study is the

development of an LC × LC system for the comprehensive analysis of endogenous

compounds in complex biological samples like urine. Urine contains many both polar,

medium polar, and relatively non-polar components, both ionic and neutral. In order to

facilitate the development of the system, a set of 32 model compounds was chosen

(Table 5.1), greatly varying in dissociation constant pKa and polarity, indicated by the

log of octanol–water partition coefficient (log P) and the log of distribution coefficient

(log D). The difference with log P is that log D is pH dependent.

The optimization of the separation of these 32 model compounds by means of LC × LC

is reported in this paper.

The need for two-dimensional separation

Obviously, the need for a two-dimensional separation system has to be demonstrated

first and its advantages proven. This is best done by performing a separation of the

test-set of 32 model compounds on a 50 mm × 1 mm I.D. reversed-phase C18 column

(3 µm particles) and an optimized flow-rate of 50 µL/min. The resulting UV

chromatogram (220 nm) is shown in 2. As can be seen from Figure 5.2 as well as from

the retention times tR and calculated resolution Rs shown in Table 5.1, several

compounds are poorly resolved or even co-eluting in the chromatogram, even

compounds that have apparently widely different log P values.


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Table 5.1: Retention times and resolutions of 32 compounds with log P and log D at pH 3 separated with a conventional LC-UV system at 220 nm

Number Compound tR (min) Rs Log P(*) Log D (**)

at pH=3

Molecular

Weight

1 Creatinine 3.83 < 0.5 -1.69 -3.3 113.12

2 Alanine 4.07 < 0.5 -3.04 -3.2 75.07

3 Dopamine 4.18 < 0.5 -0.22 -2.8 153.18

4 Aniline 4.18 < 0.5 0.94 -0.6 93.13

5 Sulfamic acid 4.21 < 0.5 -1.42 -3.4 173.19

6 Tyrosine 4.22 < 0.5 0.38 -2.2 181.21

7 Sulfaguanidine 4.53 < 0.5 -1.22 -1.3 214.24

8 3,4-Hydroxybenzoic acid 8.60 2.0 1.16 1.1 154.12

9 Tryptophan 9.63 < 0.5 1.04 -1.5 204.22

10 Paracetamol 10.02 < 0.5 NC NC 151.16

11 2,6-Dimethylanilin 15.01 2.0 NC NC 121.86

12 Theophyline 20.46 2.8 -0.18 -0.2 180.16

13 Nadolol 21.07 2.1 1.29 -1.8 309.45

14 Caffeine 22.61 1.7 -0.13 -0.1 194.19

15 Vanillin 23.48 1.8 1.19 -1.2 152.15

16 Sulfamethoxypyridazine 24.09 1.0 NC NC 280.34

17 Thiobenzamide 24.62 1.0 NC NC 137.21

18 Metamitron 24.62 < 0.5 0.83 0.8 202.21

19 Benzoic acid 24.95 < 0.5 1.87 1.9 122.12

20 Atrazin-desethyl 26.10 0.9 1.50 1.4 187.63

21 Aspirin 26.31 1.3 1.23 1.1 180.16

22 Midazolam 26.83 3.4 1.53 1.7 325.93

23 Imipramine 27.47 3.3 4.80 1.7 316.88

24 Hydrocortison 29.39 1.8 1.43 1.4 362.46

25 Carbamazepine 31.82 0.9 2.67 2.7 236.33

26 Nitrazepam 32.92 1.0 2.18 1.8 281.27

27 Prometryn 33.72 0.5 3.44 2.4 241.35

28 Oxazepam 34.21 4.8 2.31 2.3 286.71

29 Desmethyldiazepam 34.63 0.5 3.15 2.6 270.72

30 Naproxen 37.46 3.2 3.18 3.0 230.26

31 Diazepam 37.88 < 0.5 2.96 2.4 284.74

32 Ibuprofen 42.78 2.7 3.72 3.7 206.32

(*) LogP was calculated with ACDlabs 8.02

(**) LogD was calculated with ACDlabs 8.05

NC: Not Calculated by ACDlabs

Chapter 5

118

However, log P can only be accurately calculated for non-ionic compounds. As several

compounds in our test-set contain one or more ionogenic groups, they may exist in

different (ionic) forms in solution, depending on the pH. Therefore, the distribution

coefficient log D would give a more appropriate description of the complex partitioning

equilibriums. From Table 1, it may be concluded that a better correlation between

retention time and log D is achieved than between retention time and log P.

Figure 5.2: Chromatogram of the 32-component test-set on the first-

dimension column only. Gradient: 0–12 min at 100% A, 12–50 min linear gradient from 100–20% A. 5_L injection and UV-detection at 220 nm.

General design of the LC × LC system

From these experiments, it was concluded that a two-dimensional LC × LC system

would be more appropriate, although a good separation could also be achieved with a

longer column and a shallower gradient program, but only at the expense of far longer

analysis times. As the composition of urine, the sample type finally aimed at in this

research project, is far more complex than that of our test-set mixture, the

development of an LC × LC seems more successful and appropriate in the long term.

A two-dimensional LC × LC system may consist of two similar columns, operated with

the same or different mobile phases, but obviously the best performance in terms of

resolution and peak capacity can be achieved if two different types of stationary phases

and mobile phases are used. Different stationary phases and different orders of these


119

columns were tested, including RPLC, hydrophilic interaction chromatography (HILIC),

cation and anion ion-exchange chromatography (CEX and AEX), and mixed-mode ion-

exchange/reversed-phase chromatography.

Both, the RPLC × RPLC and HILIC × RPLC experiments (data not shown) did not

provide additional separation in comparison with conventional RPLC separation. The

results obtained with an RPLC × RPLC setup show similar selectivity for the compounds

in comparison with a one-dimensional setup. This can be concluded from the 2D plot

which shows that the analytes are located in a straight slope. In the HILIC × RPLC

setup, the high organic modifier gradient (99–80% of ACN) of the first-dimension

HILIC mobile phase hampered the separation on the second-dimension RPLC column.

Most test compounds did not exhibit any retention at all. An intermediate dilution step

with water resulted in unfavorable sample dilution.

Further experiments showed that a combination of RPLC and a mixed-mode CEX/RPLC

column results in the highest stationary phase orthogonality for the compounds in our

test-set.

The mixed-mode column was preferred over conventional CEX or AEX columns because

it can be operated without the need for high salt concentrations in the mobile phase,

and is therefore more readily compatible with MS detection. With respect to the

separation of highly polar and especially early-eluting ionogenic compounds, a setup

consisting of RPLC in the first dimension and the mixed-mode CEX/RPLC column in the

second dimension is the best solution: compounds poorly retained in the first-

dimension RPLC are then separated in the second dimension. When using a reversed

column order with a mixed-mode CEX/RPLC column in the first dimension and RPLC in

the second dimension, the separation of most ionogenic compounds is strongly

hampered. Since the second-dimension mixed-mode CEX/RPLC column exists of two

different separation mechanisms, two different mobile-phase optimizations are

required. Our solution was to use step gradients in the second dimension. Additionally,

the chosen setup can readily be converted into a one-dimensional RPLC system,

whenever necessary for development purposes.

The LC × LC system consisted of a 50 mm × 1 mm I.D. RPLC first-dimension column,

operated at a flow-rate of 50 µL/min and a 30 mm × 4 mm I.D. Primesep A second-

dimension column, operated at a flow-rate of 1.5 mL/min. Transfer of 60-s fraction

from the first-dimension separation to the second-dimension column is performed by

means of an electronically controlled two-position switching valve, containing two 25-

µL sample loops, which are alternatingly used for fraction collection from the first-

dimension column and injection of the previous fraction into the second-dimension

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120

column. This means that the modulation time is 60 s. The first-dimension separation is

achieved using a linear mobile-phase gradient, while the second-dimension separation

is performed isocratically. Isocratic elution is easier to handle in the fast runs (typically

60 s) in the second-dimension separation and avoids the need for a column-

conditioning step and the negative effects of the rapid changes of the modifier

concentration on the background signals of UV or MS detectors. Nevertheless, gradient

elution in the second dimension would be more favorable, because it would reduce the

number of wrap-rounds. A wrap-around occurs, when the retention time of a

component in the second-dimension separation exceeds the modulation time. In that

case, the component elutes with compounds from the next modulation cycle, at a

retention time different than its real retention time, thus possibly resulting in co-elution

problems, and with a significantly broader peak, adversely affecting the detection limit

of that component.

Injection volume in first-dimension column

In choosing the injection volume in the first-dimension column, it must be considered

that a relatively large injection volume is preferred, given the expected low

concentrations of some of the relevant endogenous compounds in urine. On the other

hand, the inner diameter of the first-dimension column, and thereby the allowable

injection volume, should be limited in order to limit the flow-rate. Complete transfer of

the column eluate of the first-dimension column to the second-dimension column,

which is operated with a typical analysis time of 60 s, is most favorable.

The influence of the injection volume, varied between 1 and 25 µL, and the analyte

retention time and peak width in the first-dimension separation was evaluated for four

compounds. The results are summarized in Table 5.2. The injection volume had little

effect on the retention time in the gradient-elution system, but significant peak

broadening (20–30%) was observed for the earlier eluting compounds at higher

injection volumes. The peak areas were not affected. An injection volume of 25 µL is

used in further experiments.


121

Table 2:

Retention time shift and peak widths related to injection volume of 4 compounds

m-Tyrosine Tryptophan Caffeine Metamitron

injection

volume

(µl)

tR

(minutes)

Width at

½ height

(minutes)

tR

(minutes)

Width at

½ height

(minutes)

tR

(minutes)

Width at

½ height

(minutes)

tR

(minutes)

Width at

½ height

(minutes)

1 2.89 0.63 6.03 0.81 9.73 0.27 11.48 0.30

5 2.86 0.68 6.00 0.86 9.70 0.27 11.43 0.31

10 2.86 0.62 6.01 0.86 9.73 0.27 11.45 0.30

25 2.74 0.80 5.80 1.06 9.68 0.27 11.39 0.31

Table 3:

Effects of eluent and the injection volume on the retention times of the re-injected fraction in the secondary column

Tryptophan(n=3) Caffeine (n=3) Metamitron(n=3) Carbamazepine(n=3) Hydrocortisone (n=3)

tR (minutes) tR (minutes) tR (minutes) tR (minutes) tR (minutes)

injection

eluent

A

84%A/

16%B

eluent

A

79%A/

21%B

eluent

A

72%A/

28%B

eluent

A

58%A/

42%B

eluent

A

62%A/

38%B

volume

(µL) 1stD eluent 1

stD eluent 1

stD eluent 1

stD eluent 1

stD eluent

25 3.85 3.53 1.09 1.04 3.21 3.05 5.61 5.37 3.94 3.64

10 3.88 3.55 1.09 1.05 3.22 3.08 5.56 5.50 3.90 3.79

5 3.87 3.52 1.10 1.06 3.21 3.09 5.12 5.08 3.92 3.82

1 3.63 3.52 1.09 1.07 3.25 3.12 5.28 5.10 3.92 3.84

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122

Effect of injection volume, organic and ionic modifier in the

second-dimension separation

The first-dimension separation is performed in gradient-elution mode. Fractions

transferred from the first-to the second-dimension column therefore differ in the

organic-modifier content. This may in turn affect the applicable transfer volume, that

is, the injection volume into the second-dimension column. This effect was studied for

five compounds, by evaluating the second-dimension retention times after direct

injection of volumes in the range between 1 and 25 µL in two solvent compositions,

that is a fixed composition of 1% acetonitrile in water with 0.1% formic acid and the

composition at which the particular compounds elutes in the acetonitrile gradient from

the first-dimension column. The results are summarized in Table 5.3. A complete

interpretation of the minor effects on retention time and peak width is complicated by

the differences in interaction of the components tested with the different retention

mechanisms active in the mixed-mode column. For instance, tryptophan shows little

volume-dependent behavior which is consistent with ion-exchange interaction, while

metamitron and hydrocortisone show more hydrophobic interaction, and is thus more

influenced by the injection volume. The most important conclusion from these

experiments is that the injection volumes, within the range of 1–25 µL, and the

mobile-phase composition have generally little influence on the retention behavior in

the second-dimension separation. This can to a large extent be attributed to the mixed

retention mechanisms in the second-dimension column.

Peak capacity of the two-dimensional system

The theoretical peak capacity of an LC × LC system can be calculated to show the

advantage of the two-dimensional LC × LC system over a one-dimension separation

system. The peak capacity nc,1 of a one-dimension separation in gradient-elution mode

can be calculated from Eq. (1) [29]:

W

ttn

RnR

c

1,,

1,

(1)

where tR,n and tR,1 are the retention times of the last and the first eluting peaks,

respectively, and W is the average 4σ peak width (at the baseline). Using this

equation, the peak capacity of the used C18 column under the conditions applied is

theoretically calculated to be 92 peaks in 46 min. The peak capacity nc,2 of the isocratic

second-dimension separation can be calculated from Eq. (2) [30]:


123

1

2,ln

41

t

tNn

n

c

(2)

where N is the plate number, and t1 and tn are the retention times of the first and the

last peak, respectively. Using this equation, the peak capacity of the mixed-mode

CEX/RPLC column is 10 peaks in 1 min.

The total peak capacity nc, tot can be calculated by multiplying the calculated peak

capacities of the two dimensions according to Eq. (3):

(3)

Thus, the total peak capacity of our system is 920 peaks. However, during loop

trapping of the 60-s fractions in the transfer step, remixing occurs of components

initially separated in the first-dimension column but contained in the same collected

fraction. Therefore, the actual peak capacity of the first-dimension column is

approximately 46 rather than 92 peaks, and consequently, the total theoretical peak

capacity of the LC × LC system under the current operating conditions can be

calculated to be 460 peaks. Although in real practice the theoretical peak capacity

cannot be obtained, the two-dimension separation obviously provides a higher peak

capacity than a conventional one-dimension separation in the same analysis time (460

vs. 92).

Optimization of the second-dimension separation

Whereas the first-dimension separation is based on hydrophobicity, the second-

dimension separation is based on both cation-exchange and hydrophobic interactions.

This implies that despite the fact that the retention mechanisms show significant

differences, the phase systems are not completely orthogonal. Whereas ACN is used as

an organic modifier in the first-dimension separation, in the second-dimension

separation both an organic modifier (ACN) and an (acidic) ionic modifier are needed for

compound elution. Formic acid, acetic acid and TFA were tested as ionic modifiers,

because these volatile acids are compatible with MS detection. However, both formic

acid and acetic acid turned out to be too weak ionic modifiers for the primesep A

column. Therefore, TFA was applied as an acidic ionic modifier in the second-dimension

separation. With this system, featuring gradient elution (1–80% ACN) in the first-

dimension RPLC column and isocratic elution (40% ACN and 0.05% TFA) in the second-

dimension mixed-mode CEX/RPLC column, reasonable good results were obtained,

2,1,, cctotcnnn

Chapter 5

124

although not completely satisfactorily because of co-elution of components in some

parts of the chromatogram. Therefore, possibilities to improve the second-dimension

separation were considered.

Gradient elution is generally applied when complex samples need to be separated

which contain compounds covering a wide range in retention factors. However, in fast

LC × LC systems, the use of gradient elution in the second dimension is not readily

accomplished due to the short modulation time (30–60 s). With only a few exceptions,

for example, when using high temperature liquid chromatography (HTLC) in the second

dimension, as presented by Stoll et al. [31], the equilibration time for reconditioning of

the second-dimension column is found to be the time-limiting factor [32]. To overcome

these limitations and still apply more optimum mobile-phase compositions in different

parts of the chromatogram, the use of step gradients can be considered. This would

require a much shorter equilibration time. However, a complicating factor related to

the use of a mixed-mode column in the second dimension is that two step gradients of

two different modifiers (ACN and TFA) might be necessary to achieve an efficient

separation.

In order to evaluate the viability and potential of step gradients in the second

dimension, a series of experiments was performed where the test-set of 32 model

compounds was analyzed with the LC × LC system with the first-dimension gradient

elution, while three different percentages of organic modifier and four different ionic

modifier conditions were applied. In these twelve experiments, ACN percentage of 20,

40, and 60% were combined with TFA concentrations of 0.05, 0.10, 0.15 and 0.20%,

respectively. Preliminary experiments showed that modifier concentrations beyond the

ranges of 20–60% ACN and 0.05–0.20% TFA resulted in excessive broad peaks or

(almost) complete loss of separation. As expected, the results showed that under

different conditions different parts of the 2D chromatogram provided better peak

resolution. From an evaluation of the number of separated peaks and the peak

resolution in the second dimension, optimum separation conditions were selected. The

peak-to-peak resolution at half heights was calculated from Eq. (4) [33]:

21 2/12/1

1218.1

ww

ttR

RR

s

(4)

where tR2 and tR1 are the retention times of adjacent peaks 2 and 1, respectively, and

w1/21 and w1/22 are the peak width at ½ height of these peaks, respectively. The

results are summarized in Table 5.4: for each of the twelve experiments the number of


125

peaks detected as well as the number of peak pairs showing a peak-to-peak resolution

better than 1.5 are given at three different time points during the LC × LC run. The

first fraction was 3–5 min, the second 27–31 min, and the third 33–37 min. The goal of

these experiments was to find second-dimension mobile-phase conditions that provide

optimum resolution in each part of the selected time windows. In some cases, that is

when the first-dimension peak was divided over two fractions, two peaks of the same

compound might be observed in two second-dimension chromatograms. These were

counted as two peaks in a time fraction. In addition, the occurrence of wraparound

peaks adversely affects the resolution in the time window. Typical baseline peak widths

in the second dimension are 6–8 s, while peaks with widths exceeding 15 s are

probably due to wrap-arounds.

Table 5.4: Number of detected peaks and resolutions in the 2nd dimension slices

Experimental conditions Fraction(1) 3-5

minutes

Fraction(2) 27-31

minutes

Fraction(3) 33-37

minutes

Experiment %TFA/%

ACN

# of

peaks

Rs>1.5 # of

peaks

Rs>1.5 # of

peaks

Rs>1.5

1 0.05/20 5 3 (A) 6 6 2 2

2 0.05/40 7 5 (B) 11 6 5 5

3 0.05/60 4 3 (C) 7 4 8 5

4 0.10/20 4 2 9 8 (D) 3 2

5 0.10/40 4 2 10 7 7 4

6 0.10/60 4 2 9 4 8 7 (G)

7 0.15/20 5 2 10 9 (E) 2 2

8 0.15/40 4 2 8 5 10 9 (H)

9 0.15/60 3 0 8 6 7 6

10 0.20/20 6 2 9 8 (F) 4 4

11 0.20/40 3 2 8 4 9 7 (I)

12 0.20/60 2 0 9 6 6 4

For each of the three fractions, the 2D chromatogram obtained under the three best

conditions in terms of the number of peaks detected and number of peaks with a

resolution better than 1.5 were studied in some more detail. The chromatograms are

shown in Figure 5.3, where Figure 5.3A, B and C correspond to the three best results

for fraction 1 (3–5 min), Figure 5.3D, E, and F with fraction 2 (27–31 min), and Figure

5.3G, H and I with fraction 3 (33–37 min) (see also Table 5.4).

Chapter 5

126

For the separation of fraction 1, representing compounds that hardly show any

retention in the first-dimension RPLC column, 0.05% TFA seems sufficient, but in fact

20% of ACN (Figure 5.3A) is not sufficient to elute these compounds without wrap-

arounds. With 60% of ACN (Figure 5.3C), on the other hand, the compounds co-elute

and show poor resolution. Therefore, a mobile phase with 40% of ACN and 0.05% of

TFA (Figure 5.3B) provides optimum results with seven peaks in one 60-s fraction and

with five peaks with a resolution above 1.5. Furthermore, the strength of the ionic

properties of this mixed-mode column is proven; even with 40% ACN the compounds

are retained.

The chromatograms of fraction 2 (Figure 5.3D, E, and F) show high similarity. The fact

that the mobile phase with 20% ACN and 0.15% TFA gave ten peaks and nine peaks

with a resolution above 1.5 indicates that probably only one peak is split over two

fractions. A somewhat closer look at the data indicates that in this fraction a low

percentage of both ACN and TFA provide the best separation.

In fraction 3 (Figure 5.3G, H, and I), both TFA and ACN play an important role in the

separation. A mobile phase with 40% ACN and 0.15% TFA (Figure 5.3H) resulted in the

highest number of separated peaks and highest number of peaks with a resolution

above 1.5.

The results of this series of experiments indicate, that optimum performance of the

second-dimension separation can be achieved by an independent and stepwise change

in the %TFA and %ACN in the mobile phase applied, while maintaining isocratic

conditions in each individual chromatogram. This will result in a higher peak capacity of

the entire LC × LC system. In addition, the number of changes in mobile-phase

composition during the LC × LC run must be limited in order to avoid excessive

background shifts which may hinder the construction of the 2D chromatograms (see

below). For instance, given the limited effect of %TFA in fraction 2 and the optimum

%TFA in fraction 3, a %TFA of 0.15% is kept throughout final part of the

chromatogram. Limiting the number of changes in the mobile-phase composition in the

second-dimension separation will also improve the reliability of the complete system.

From these experiments, it may be concluded that the conditions applied for Figure

5.3B, E and H can be considered optimum for the test-set of 32 components. The 2D

chromatograms from complete LC × LC runs acquired using either of these conditions

which are depicted in Figure 5.4. These chromatograms indicate, that optimum

separation can be achieved by the use of three different mobile-phase compositions for

the second-dimension separation, that is 0.05% TFA and 40% ACN during the first 12

min of the first-dimension separation, 0.15% TFA and 20% ACN between 12 and 31


127

min, and 0.15% TFA and 40% ACN between 31 and 50 min of the first-dimension

separation.

Figure 5.3: Expanded 2D chromatograms of three different time windows obtained under different isocratic solvent conditions in the second-dimension separation. Gradient elution in the first-dimension separation (see Figure 5.2 for conditions). Fraction 1 (timer window 3–5 min): (A) 0.05% TFA, 20% ACN, (B) 0.05% TFA, 40% ACN, (C) 0.05% TFA, 60% ACN. Fraction 2 (time window 27–31 min): (D) 0.10% TFA, 20% ACN, (E) 0.15% TFA, 20% ACN, (F) 0.20% TFA, 20% ACN. Fraction 3 (time window 33–37 min): (G) 0.10% TFA, 60% ACN, (H) 0.15% TFA, 40% ACN, (I) 0.20% TFA, 40% ACN.

Figure 5.4: 2D chromatograms of the 32-component test-set obtained with gradient elution in the first-dimension separation (see Figure 5.2 for conditions) and with isocratic elution under the three different optimal conditions in the second-dimension separation: (A) 0.05% TFA, 40% ACN; (B) 0.15% TFA, 20% ACN; (C) 0.15% TFA, 40% ACN.

Chapter 5

128

These time windows are shown as boxes in the chromatograms in Figure 5.4. Note that

serious co-elution and wrap-arounds occur outside these boxes in either of the 2D

chromatograms in Figure 5.4.

Subsequently, these optimized conditions were applied within one LC × LC run. A

typical chromatogram obtained is shown in Figure 5.5. The data show that good

separation is obtained for most of the 32 components in our test mixture. The data

also clearly show the effect of the step gradient, especially of the change in TFA

concentration on the construction of the 2D chromatogram: the higher TFA

concentration in the second and third time zone results in a more significant UV cut-off,

represented by a color change of the drawing of the background.

Figure 5.5: 2D chromatograms of the 32-component test-set obtained with gradient elution in the first-dimension separation (see Figure 5.2 for conditions) and with the three step-gradient isocratic elution in the second-dimension separation: time window 1–12 min: 0.05% TFA, 40% ACN; time window 12–31 min: 0.15% TFA, 20% ACN; and time window 31–50 min 0.15% TFA, 40% ACN

Under these optimized conditions, repeatability and reproducibility of the retention

times in the chromatograms were tested. The intraday (n = 3) and interday (n = 4)

variability of the retention times (expressed as RSD) was 0.5% and 0.7%, respectively.

Good linearity (R2 higher than 0.996) was also achieved when acquiring calibration


129

curves in the concentration range between 5 and 100 µM for three different compounds

varying in polarity, that is caffeine, metamitron and hydrocortisone. Detection limits of

caffeine, metamitron and hydrocortisone were between 1 and 2 µM.

Combining LC × LC with APCI-MS detection

When the LC × LC system developed will be applied to the separation of endogenous

compounds in urine, for instance, for metabolomics or biomarker studies, the use of

MS detection rather than UV detection will be an important aspect. Therefore, in

developing the mobile-phase conditions for the LC × LC system, mobile-phase

compatibility issues were already taken into account: no non-volatile mobile-phase

additives were considered. However, the use of TFA was found to be important in the

efficient use of the second-dimension mixed-

Figure 14: 2D chromatograms of the 32-component test-set with (A) UV-detection at 220 nm, and (B) total-ion current of positive-ion APCI-MS detection. Compounds: 1, 5–7, 9–18, 20–21, 24–32 were identified. Compound numbers are corresponding with the compounds in Table 5.1. Condition: see Figure 5.5 and Section 2.

mode CEX/RPLC column, as both formic and acetic acid turned out to be too weak ionic

modifiers. The presence of TFA in the mobile phase as well as the high flow-rate of the

second-dimension column, that is typically 1.5 mL/min, indicates that the use of APCI

in positive-ion mode for analyte ionization in MS is most likely more appropriate than

the use of electrospray ionization (ESI). TFA is known to provide ionization suppression

effects in ESI and high flow-rates are not optimum for ESI either. Figure 5.6 shows the

2D chromatogram of LC × LC separation with the optimized conditions using UV and

MS detection simultaneously. A combination of the two detectors is favorable because

some compounds do not ionize under these conditions, while some compounds do not

show UV activity. Compound 30 and 32 (naproxen and ibuprofen), for instance, are

readily detected by UV detection (Figure 5.6A), but are not detected by positive-ion

APCI-MS (Figure 5.6B), whereas compounds 29 and 31 (desmethyldiazepam and

Chapter 5

130

daizepam) are only weakly detected in the UV chromatogram (Figure 5.6A), while they

clearly show up in the APCI-MS chromatogram (Figure 5.6B). By the use of both UV

and MS detection, at least 26 compounds of 32 components can be detected and

quantified. The peaks of compounds 22 and 31 are relative broad, which indicates that

these compounds are wrapped around. This has to be taken in account if these

compounds have related to a peak in the first-dimension analysis.

Figure 5.7: 2D chromatograms of (A) a four-time diluted human urine sample, and (B) the same four-time diluted urine sample spiked with 25 µM of the 32-component test-set. Compounds identified by APCI-MS, (7) sulfaguanidine; (1) creatinine; (14) caffeine; (24) hydrocortisone; (25) carbamazepine; (28) oxazepam; (31) diazepam. Condition: see Figure 5.5 and Section 2.

As a final step in the developmental project for comprehensive two-dimensional LC ×

LC, four-time diluted urine samples, with and without spiking with the test-set of 32

components, were analyzed with the complete system, featuring both UV and positive-

ion APCI-MS detection. The resulting chromatograms are shown in Figure 5.7. No

attempts were made to identify the various endogenous components that showed up in

the urine sample, although both creatinine and caffeine were readily recognized. A

number of the components from the test-set detected in the chromatogram of the

spiked urine sample are also indicated in Figure 5.7B. These data show the potential of

the developed technology in urine analysis.


131

Conclusion

In this paper, the development and optimization of a comprehensive two-dimensional

LC × LC system was described. The system features a 60-min gradient elution on a

first-dimension RPLC column, subsequent transfer of the column eluate in 60-s

fractions, and isocratic elution based on a step gradient of both organic and ionic

modifier (ACN and TFA) on a second-dimension mixed-mode cation-IEC/RPLC column.

The multi-step isocratic elution in the second dimension resulted in a higher peak

capacity and improved peak resolution of the complete system, without compromising

stability and background of the detectors used. Simultaneous UV and APCI-MS detec-

tion was demonstrated, which expands the applicability range of this approach in the

analysis of complex mixtures. Development and testing of the system was performed

using a 32-component test mixture of compounds widely differing in physico-chemical

properties (pKa, polarity measured as log D, ranging from −3.4 to +3.7). Some

preliminary results with (spiked) urine samples were also obtained.

The final system is highly flexible and has a broad applicability. The changes of the

organic and ionic modifier concentrations in the multi-step program are readily adapted

to optimize separation and peak capacity for other or even more complex mixtures.

The experimental strategy outlined can be applied in the optimization of two-

dimensional separation of other such mixtures. The compatibility with both UV and MS

detection provides chromatographic information with a high information content

concerning the sample constituents.

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