It is possible to recognize the molecule type from its mass and the kind of ions that its fragmentation produce.

It is possible to recognize the molecule type from its mass and the kind of ions that its fragmentation produce.

We are going to give a brief explanation of how do these machines work, their technical functioning.

4.1. UPLC™ Analysis

Before start describing the technical data’s and the experimental conditions that we use in our research, it is necessary to introduce the physical guide principles which drive a liquid chromatography and the parameters which influence the separation.

The importance of “cleaning” a chromatogram is quite deducible, the more the peaks are separated the easier will be further analysis and compound detecting, for example MS/MS analysis.

In this work we will examinate, giving some clues of theory, which are the main parameters that influence a liquid chromatography separation, trying to understand the inter-connections between some of them, and changing their values.

Analysing the results to obtain the best chromatogram by peak resolution and number, we had the possibility to understand which the best values for the several technical parameters were.

These parameters have been used to obtain insight into the process of monolignol coupling in the cell wall by characterizing the chemical structures of a large number of molecules by UPLC™ and mass spectrometer analysis.

High-performance liquid chromatography (HPLC) is a form of column

chromatography that is used to separate components of a mixture by using differential

chemical interactions between the several compounds being analyzed and the

chromatography column. In a HPLC separation, a metabolite is forced, by pumping a high

pressure liquid (mobile phase), through a column called stationary phase. A column is a

tube packed with small round particles with a certain surface chemistry. The sample is

introduced in a small volume to the stream of the mobile phase and its’ molecules are

retarded by specific chemical or physical interactions with the stationary phase as they

traverse the length of the column. The amount of retardation depends on the nature of the

analyte, stationary phase and mobile phase composition. The time at which a specific

compound elutes (comes out from the end of the column) is called retention time and is characteristic of a given metabolite.

Figure 21

Simplified scheme of a HPLC chromatographer. A pump forces the mobile phase through the system in which the sample is introduced. A column performs the separation of the different molecules that are identified by a detector. At last, data’s can be raised and elaborated by a computer.

Reversed phase HPLC (RP-HPLC) is a particular kind of HPLC separation, characterized by a non-polar stationary phase and a polar mobile phase. One common stationary phase is silica particles on which surface are linked chains of alkyl groups such as C

H

or C

H

.

Retention time is therefore longer for more non-polar molecules, allowing polar molecules to elute faster. RP-HPLC operates on the principle of hydrophobic or hydrophilic interactions between the metabolite and the alkyl chains linked to the silica particles. The characteristics of the molecule play an important role in its retention. In general, an analyte with a longer alkyl chain length results in a longer retention time because it increases the molecule's hydrophobicity, on the contrary a polar molecule will have a shorter retention time.

Whether the isocratic separation maintains a constant composition of the mobile

phase, during the gradient elution is possible to vary it. For example, a normal gradient for

reverse phase chromatography might start at 1% acetonitrile (ACN) and progress linearly

to 70% in 40 minutes. Time and solvent ratio can be varied in relation with the solvent type

and the metabolites being searched. Gradient separates metabolites mixture as a function of

Figure 22

Detail of the stationary phase’s silica particle.

Molecules eluting through the column interact with the alkyl groups chains. Separation is due to a differential elution based on the polarity of the molecules.

One compound can have a higher affinity for the stationary phase with a particular mobile phase and will present a higher retention time than another metabolite with a lower affinity for the column and a higher one for the mobile phase. The application of HPLC to biochemical samples today is widespread. It can be used for product purification or in our case to detect particular molecules, which compose the lignin of transgenic plants. In the last years, progresses of technology have brought more precise and faster HPLC machine.

UPLC

stands for ultra-high performance liquid chromatography and it is an evolution of the HPLC technology. The greatest difference between the 2 systems is the higher pressure reachable from the UPLC

system. A pressure increasing permits to:

• reduce the particles size of the stationary phase

• use longer column

As a consequence, the efficiency of the separation increases, better separated peaks can be detected. In particular, particle diameter plays an important role on the efficiency as it influences the resistance to mass transfer (C’ term of the Van Deemter equation, see pg.3). Little particles present little channels in which molecules can flow. For a molecule is

Particle surface

Alckyl group chains

Mobile phase Particle

surface

Alckyl group chains

Mobile phase

easier flowing out of from a particle channel if it is shorter. For this reason, resistance to mass transfer will be lower likewise the C’ term of the Van Deemter equation. Decreasing of the C’ term causes the same results on the H term, but, according to N=L/H equation, an increasing of the efficiency. At last, a higher pressure of injection of the samples through the mobile phase and a smaller dimension of the column’s particles produce narrower peaks and an increasing of their resolution.

We will not go into details on what the theory of UPLC

separation is, but we will try to have a general view on what are the basis of it and the parameters on which we can play on.

The separation of any two bands in the chromatogram can be varied systematically by changing experimental conditions.

Resolution R

can be expressed in terms of three parameters (k, α, and N), which are directly related to experimental conditions:

k is the average retention factor for the two bands (formerly referred to as the capacity factor k’), N is the column plate number, and α is the separation factor; α=k

/k

, where k

and k

are values of k for adjacent bands 1 and 2. The equation above is useful in method development because it classifies the dozen or so experimental variables into three categories: retention (k), column (N), and selectivity (α).

R

= ¼ (α-1) N

k/(1+k)

Figure 23

Effect of k, N and α on resolution

The first parameter that has to be optimized is the retention (k). The value of k depends on the equation k= 20/G

where G

is the gradient steepness.

G

= (V

∆ % B)/(t

F)

V

is the dead column volume and it is constant unless the column is changed.

∆%B is the increase of %B (solvent composition) during the time t

F is the flow of the mobile phase

t

is the time gradient.

As we can see k can be varied playing on some values.

Likewise, selectivity (α) is directly linked to k because it is the ratio between 2 retention values and it depends on solvent type, buffer type, stationary phase, temperature. Changes in k also affect ∆.

Initial separation

Vary k

Vary N

Varyα

tim e Initial separation

Vary k

Vary N

Varyα

tim e

α =k

/k

The last parameter that we consider is the efficiency(N). Efficiency depends on plate height (H) and column length (L) according to the equation:

where L is a constant value depending on the type of column while H can vary with the velocity u of the mobile phase as it passes through the column.

The value of H is explained by the Van Deemter equation:

A’ is the Eddy diffusion, which represents the band broadening due to different

path length that the molecules can choose. This term is particle size dependent and u

independent. The smaller the particles are, the more obliged will be the path to flow out of

the column and of course the less band spreading there will be.

Figure 24

Eddy diffusion’s representation.

Bigger particles offer a larger choice of paths for the molecules to elute through the column. There will be a slightly difference of the molecules’ detection time (band broadening).

B’ term belongs to the longitudinal diffusion, the phenomenon for which molecules spread out in the mobile phase. With a high speed (u) of the mobile phase the molecules move quicker in the column and they are fewer influences by the diffusion.

INJECTION OF THE SAMPLE

DIFFUSION

FLOW

FLOW Low flow

FLOW High flow

DETECTOR INJECTION OF THE SAMPLE

DIFFUSION

FLOW

INJECTION OF THE SAMPLE

DIFFUSION

FLOW

DIFFUSION

FLOW

FLOW FLOW Low flow

FLOW High flow

DETECTOR

Figure 25

Longitudinal diffusion’s representation.

A higher flow permits to the molecules to avoid the molecules “spreading out” in the mobile phase. As a consequence, correspondent peak presents a better resolution.

C’ term is called resistance to mass transfer for which mobile phase stagnates in particle pores. Molecules of the samples that enter a pore are only subjected to diffusion and it takes time before they leave the stationary phase or they can remain stuck on it.

Solutions to avoid these problems are:

Keep u (velocity of the mobile phase) low Narrow particles

Use low-viscosity solvents

FLOW

COLUMN

TAKES TIME BEFORE MOLECULES LEAVE THE PORES

LONG PORE

DETECTOR FLOW

COLUMN FLOW

COLUMN

TAKES TIME BEFORE MOLECULES LEAVE THE PORES

LONG PORE

DETECTOR

Figure 26

Resistance to mass transfer’s representation.

Small particle with a small diameter permits a faster elution of the molecules out of the particle’s pores. As a consequence, resultant peak’s resolution will increase.

Notice that while in the B’ term of the Van Deemter equation flow needs to be high, in the C’ term better results can be reached if the flow is slow. U (velocity of the mobile phase) has to be high enough to avoid molecules spreading out, but not too high to don’t influence C’ term in a negative way. Further explanation can be obtained in the figure below.

Figure 27

The ideal velocity of the mobile phase depends on B’ and C’ terms.

As you noticed above, lots of parameters can be changed to perform the best

separation. It is almost impossible to optimize all of them because most of them are linked

to each other. For this reason, a changing of one parameter can worsen the separation

because other parameters can be linked to that one or vice versa. In conclusion, in our

experience, we varied technical values starting from old HPLC parameters.Analyzing the results, in therms of peaks number and their resolution, it has been possible to decide which were the best values to be used for following experiences.

4.2. Mass Spectrometer (MS)

Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of ions. It is most generally used to find the composition of a physical sample by generating a mass spectrum representing the masses of sample components. Different chemicals have different masses, and this fact is used in a mass spectrometer to determine what chemicals are present in a sample. The mass spectrum is measured by a mass spectrometer.Mass spectrometers consist of three basic parts: an ion source, a mass analyzer, and a detector system. The stages within the mass spectrometer are:

1. Production of ions from the sample 2. Separation of ions with different masses

3. Detection of the number of ions of each mass produced 4. Collection of data to generate the mass spectrum

A schematic of the basic set up of a mass analyser is shown in

. The

ions, produced in the source of the instrument, enter into the trap through the inlet and are

trapped through action of the three hyperbolic electrodes: the ring electrode and the

entrance and exit endcap electrodes. Various voltages are applied to these electrodes which

results in the formation of a cavity in which ions are trapped. The ring electrode RF

potential, an a.c. potential of constant frequency but variable amplitude, produces a 3D

quadrupolar potential field within the trap. This traps the ions in a stable oscillating

trajectory. The exact motion of the ions is dependent on the voltages applied and their

individual mass-to-charge (m/z) ratios. For detection of the ions, the potentials are altered

to destabilise the ion motions resulting in ejection of the ions through the exit endcap. The

ions are usually ejected in order of increasing m/z by a gradual change in the potentials.

Figure 28

A Schematic view of a Quadrupole Ion Trap Mass Analyser.

The underlying principle of all mass spectrometers is that the paths of gas phase ions in electric and magnetic fields are dependent on their mass-to-charge ratios which is used by the analyzer to distinguish the ions from one another.

The mass spectrometry technique is applicable in:

•

identifying unknown compounds by the mass of the compound molecules or their fragments

•

determining the isotopic composition of elements in a compound

•

determining the structure of a compound by observing its fragmentation

•

quantifying the amount of a compound in a sample using carefully designed methods (mass spectrometry is not inherently quantitative)

•

studying the fundamentals of gas phase ion chemistry (the chemistry of ions and neutrals in vacuum)

•

determining other physical, chemical, or even biological properties

of compounds with a variety of other approaches

5. EXPERIMENTAL WORK

At the beginning, we deeply worked on a new liquid chromatography machine (UPLC™) setting the parameters which permit an optimal separation of the molecules.

This work has been deeply analyzed above in the UPLC™ theory, explaining which are the several parameter which can be varied. In the results we present, step by step, the optimization of the several parameters value from the comparison between the peak number and resolution in the various conditions. In the end, we also focused our attention on the trials we performed to obtain a clear separation of the peaks which compose a chromatogram. This operation took us a lot of time but these technical parameters are now used from the research group.

In the second part of my experience at the VIB, once the machine was ready to perform experiments, we started focusing on the research project.

Our aim was to obtain insight into the process of oligolignol biosynthesis in the cell wall of genetically modified Arabidopsis thaliana stems by using UPLC

- MS/MS analysis.

These plants have been processed by up-regulating ferulate-5-hydroxylase (F5H) and downregulating caffeic acid O-methyltransferase (COMT) genes obtained with the procedures seen on chapter

The study of this plant phenolic fraction is important to better understand lignin polymerization and deposition and to answer some pertinent questions about monolignol coupling in vivo. To identify this oligolignol fraction, we profiled the methanol-soluble phenolics present in xylem extracts of genetically modified Arabidopsis thaliana plants.

MATERIALS AND METHODS

Arabidopsis Thaliana engineered seeds presents:

•

Wild-type and F5H up-regulated/COMT down-regulated Arabidopsis thaliana seeds were set in petri dishes with a specific growing medium. According to Mendel laws, only 1 seed out of 4 was recessive for COMT gene.

Growing medium protocol (20 dishes/l).

4,3 gr. Murashige and Skoog medium (Duchefa, Haarlem, The Netherlands).

10 gr. Sucrose

0,5 gr. 2-(N-morpholino)ethanesulfonic acid (MES) adjust pH to 5.7 with KOH

7,25 gr. Agar

X

CCOMT ccomt

heterozygous

Self-pollination

Recessive Ho Dominant Ho

He

He

Autoclavation

1 ml. Vitamin solution

To perform the experiment we divided the dishes, with the same growing medium, in 2 groups respectively WT (wild type) and DM (double mutants). Each dish contain 64 positions in which seeds could be be set. The dishes have been manteined under 16-hours light/8-hours dark photoperiod at 100 µE٠m

٠s

at 22°C in the greenhouse for 3 weeks.

In DM dishes, there was the contemporary presence of CCOMT homozygous ad heterozygous plants.

Homozygous and heterozygous plants can not be recognized unless they reach a certain dimension.

In fact, heterozygous plants grow faster than the homozygous so they can be

separate after 3 weeks of growth.

After three weeks of growth homozygous plants, characterized by small dimension were transferred to plastic boxes filled with the same growing medium to permit the best root, stem and leaves development.

Arabidopsis plants have been for 2 months until harvest, reaching a height of

approximately 10 cm.

Plant stem were harvested, eliminating roots and leaves and then processed for lignin extraction.

Stems have been grinded in a mortar using a pestle and liquid nitrogen.

Thanks to its low temperature, liquid nitrogen crystallizes the stems easing the grinding operation. After grinding in liquid nitrogen, the stems are reduced into powder and the oligolignol extracting protocol can now be applied. In an eppendorf we added the grinded sample and the chemicals to perform the extracting.

• Add 1,5 ml MeOH to break the cell wall

• Spin down, for 5 min. at 14.000 rpm

• Remove MeOH from the eppendorf

• Dry MeOH fraction in the SpeedVac

• Resolve dried pellet in cyclohexane/water (1,5 ml each)

• Remove water (containing the oligolignols) and put in a vial to

perform the UPLC™ injection

LC-MS Analysis

UPLC™ fractions were injected by means of a Waters Acquity UPLC™

autosampler onto a reversed-phase Waters Acquity UPLC™ column 2.1 x 150 mm and 2.1 x 100 mm., particle size 1,7 µm., pore diameter 180 Å (18 nm.). A gradient separation (Waters UPLC™ pump) was run from (water 99%, 1% acetonitrile, 0.1% buffer; solvent A, pH 3) to acetonitrile 99% 1% H

O ; 0.1% buffer; solvent B, pH 3) varying the following conditions: flow, column temperature, time, gradient, buffer type. A SpectraSystem UV6000LP detector (Thermo Separation Products) measured UV/V absorption between 200 and 450nm with a scan rate of 2 scans/s.

Atmospheric pressure chemical ionization, operated in the negative ionization mode, was used as an ion source to couple UPLC

with an MS instrument (LCQ Classic;

ThermoQuest, San Jose, CA; vaporizer temperature 450°C, capillary temperature 150°C, source current 5 µA, sheath gas flow 21, aux gas flow 3).

During separation, the most abundant ion in each full MS scan was fragmented in the next scan with the dependent MS/MS mode. Additionally, each fraction was separated on LC-MS/MS under higher acidity buffer conditions.

6. RESULTS

In our experiments, we performed several separations, varying one parameter or more every run. We wanted to know which were the best gradient curve and the best flow for our separation. We also tested 2 different columns of different length: 10 cm. Vs. 15 cm. Theoretically, the longer the column is, the better the separation will be. An explanation of this fact can be found on the equation N= L/H; the more the column is long, the more the efficiency is elevated. Gradient has been tested using 2 kind of gradient curve, respectively linear and “exponential shape” (

A and B).

The gradient and the experimental conditions are shown in the following tables.