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Protocols

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PCR

Aim
The purpose of a PCR is to amplify DNA fragments from primers specific to the latter.

Materials

• Thermocycler

• PCR Tubes, 0.2 ou 0.5 mL

• DNA

• dNTP

• Buffer 10x

• Taq polymerase QIAGEN

• MgCl2

• Primer Forward

• Primer Reverse

• H2O RNAse Free

Procedure
Using the Taq polymerase from QIAGEN

Perform a PCR mix according to the following conditions:

Reagents	Quantity (µL)
Buffer 10x containing MgCl2	2.5
dNTP (20µM)	0.5
Primer Forward (0.5µM)	1.25
Primer Reverse (0.5µM)	1.25
ADN (<1µg)	Variable
Taq polymerase QIAGEN (2,5u)	0.5
H20	q.s.p 25 µL

The PCR is then started according to the following conditions:

First denaturation 94°C	94°C	4 min
Denaturation	94°C	2 min
Hybridation	Variable, depending on Primer Tm	30 sec - 1 min
Elongation	72°C	2 min
Elongation	72°C	10 min

35 cycles are performed from Denaturation to Elongation.

The PCR products can then be analyzed on gel with the addition of a loading buffer or purified, or digested.

OR
Using the Taq polymerase from QIAGEN

Perform a PCR mix according to the following conditions:

Reagents	Quantity (µL)
5X Green GoTaq Reaction Buffer	2.5
dNTP (10 mM)	0.5
Primer Forward (10 µM)	1.25
Primer Reverse (10 µM)	1.25
ADN (<0,5µg/50µL)	Variable
"GoTaq DNA polymerase PROMEGA
(5 u/µL)"	0.5
H20	q.s.p 25 µL

The PCR is then started according to the following conditions:

First denaturation 94°C	95°C	2 min
Denaturation	95°C	1 min
Hybridation	Variable, depending on Primer Tm	1 min
Elongation	72°C	1 min/kb
Elongation	72°C	10 min

35 cycles are performed from Denaturation to Elongation.

The PCR products can then be analyzed on gel with the addition of a loading buffer or purified, or digested.

Purification of PCR products

Aim
The purpose of purifying the PCR products is to remove reagents from the mix and concentrate the DNA in buffer or water.

Materials

• QIAquick PCR & Gel Cleanup Kit (Ref 28506)

• Tube 1,5 mL

• Centrifuge at 13,000 rpm

Procedure

Add 5 volumes Buffer PB to 1 volume of the PCR reaction and mix.
Place a QIAquick column in a 2 mL tube.
To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min.
Discard the flow-through and place the QIAquick column back in the same tube.
To wash, add 750 µL Buffer PE to the QIAquick column and centrifuge for 1 min.
Discard flow-through and place the QIAquick column back into the same tube.
Centrifuge the column for 1 min to remove residual wash buffer.
Place each column in a clean microcentrifuge tube.
To elute DNA, add 50 µL of EB Buffer (10 nM Tris-Cl, pH 8.5) or water to the center of the membrane and centrifuge, let 3 min at room temperature then centrifuge for 2 min.
Load a DNA sample with loading buffer to analyze the PCR purification on agarose gel and / or store the DNA at -20°C.

Digestion

Aim
Digestion of DNA fragments and vectors creates cohesive ends between the two to assemble them in a ligation protocol.

Materials

• Restriction enzyme

• Buffer

• BSA

• H20 free RNAse

• Insert

• Linearized plasmid

• Tube 1,5 mL

• Stove at 37°C

• Heatblock 80°C

Procedure

Make a digestion mix as follows:

Reagents	Quantity (µL)
Buffer 10x	2
BSA	0.5
Restriction Enzyme 1	0.5
Restriction Enzyme 2	0.5
H20	q.s.p 20 µL

Mix 4 μL of digestion mix with 4 μL of insert or vector.
Incubate at 37°C for 1 hour.
Inactivate the enzyme by incubation at 80 °C for 20 min.
Digestion products are ready for ligation. Store at -20°C.

Ligation

Aim
The purpose of the ligation is to fold a DNA sequence inside a vector in order to transform competent bacteria.

Materials

• Digested linearized plasmid

• Digested purified insert

• Buffer 10x

• T4 DNA Ligase

• Distilled water

• Heat Block 80°C

Procedure

Mix the digested insert with digested linear plasmid in a 3:1 mole ratio thanks to the following formula:

Add 1 μL of 10x buffer and 0.5 μL of T4 DNA Ligase.
Add water RNAse free q.s.p 10 µL.
Mix and let stand at room temperature overnight at 4°C.
Inactivate the ligase by heating the tube for 10 min at 65°C.
A transformation can then be performed.

Transformation

Aim
A bacterial transformation aims to insert a plasmid containing a selected DNA fragment in a bacterium to change its phenotype.

Materials

• Competent Cells

• LB medium

• Ice

• Bath at 42°C

• Ligation product

• Agitator incubator at 37°C, 225 rpm

• LB agar with adequate antibiotic

• Stove at 37°C

Procedure

Thaw competent cells on ice.
Mix 2 μL of ligation product with 25 μL of competent cells and let stand on ice 30 min.
Achieve a thermal chox for 45 sec at 42°C.
Place immediately the cells on ice for 2 min.
Add 975 µL of liquid LB medium without antibiotics.
Incubate cells at 37°C with 225 rpm for 1 hour.
Spread the cells on Petri dishes of LB agar in proportion 1/10th and 9/10th in volume and place them in the oven overnight.

Colony PCR

Aim
The objective of a colony PCR is to check the clones obtained after transformation. This ensures that the integrated plasmids are properly constructed in bacteria.

Materials

• Thermocycler

• PCR Tubes, 0.2 ou 0.5 mL

• Bacterial colony

• dNTP

• Buffer 10x

• Taq polymerase

• MgCl2

• Primer Forward in insert

• Primer Reverse in plasmid

• H2O RNAse free

Procedure

Make a mix as described in the protocol (previously described in the PCR part) without putting DNA.
Collect a colony from the agar using a tip and immerse the tip in the PCR mix.
Colony PCR can be performed on as many clones as you like. You can return the selected clones in liquid culture without forgetting to add the appropriate antibiotic in the liquid culture medium.
The PCR is then started according to the same conditions as previously described.
The PCR products can then be analyzed on gel with the addition of a loading buffer if needed.

Minipreparation

Aim
The goal of a mini-preparation is to recover the plasmid DNA present inside the bacteria in liquid culture.

Materials

• QIAprep Spin Miniprep kit (Ref 27106)

• Centrifuge

• Tube 1,5 mL

Procedure

Centrifuge the bacterial cells at 8000 rpm for 3 min at room temperature.
Resuspend the pellet in 250 µL in P1 buffer.
Add 250 µL of P2 buffer and mix gently by inverting the tube.
Add 350 µL of N3 buffer and centrifuge at 13000 rpm for 10 min.
Apply 800 µL of the supernatant to the QIAprep 2.0 spin column by pipetting.
Centrifuge 1 min at 13000 rpm and discard the flow-through.
Wash the QIAprep 2.0 spin column by adding 500 mL Buffer PB and centrifuging for 1 min. Discard the flow-through.
Wash the QIAprep 2.0 spin column by adding 750 Buffer PE and centrifuging for 1 min at 13000 rpm.
Discard the flow-through, and centrifuge 2 times at full speed for an additional 1 min to remove residual wash buffer.
Place the QIAprep 2.0 column in a clean 1.5 mL microcentrifuge tube. To elute DNA, add 50 μL Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep 2.0 spin column, let stand for at least 1 min and centrifuge for 1 min.
Load a DNA sample with loading buffer to analyze the miniprep products on agarose gel and / or store the DNA at -20°C.

Enzymatic digestion control

Aim
The control of the clones obtained by a transformation can also be performed by enzymatic digestion. This allows once again to verify that the bacteria have integrated properly constructed plasmids.

Materials

• Restriction enzymes

• Buffer 10x

• BSA

• Free RNAse water

• Stove 37°C

• Heatblock 80°C

• Tube 1,5 mL

• DNA

Procedure

Make a minipreparation (Refer to Minipreparation protocol) of the clones that you wish to test.
Make a digestion mix as follows.
The restriction enzymes must be chosen to generate fragments of different sizes. To ensure the proper integration of the insert in the plasmid, it is advisable to choose an enzyme that has a site inside the insert and on the plasmid.

Reagents	Quantity (µL)
Buffer 10x	2
BSA	0.5
Restriction Enzyme 1	0.5
Restriction Enzyme 2	0.5
H20	q.s.p 20 µL

Mix 4 μL of digestion mix with 4 μL of miniprep product.
Incubate at 37°C for 1 hour.
Inactivate the enzyme by incubation at 80°C for 20 min.
Digestion products are ready for analyse adding loading buffer and loading the samples on agarose gel.

Bacterial inclusion in collection

Aim
The aim of the inclusion in collection is to keep the bacteria that we have as it is, without any modification in its genome, for at least 20 years.

Materials

• Bacterial strain

• LB medium

• Antibiotics

• Glycerol 99%

• Cryotube

Procedure

Make a culture medium by adding LB medium + the antibiotics the strain resists to.
Add 25µL of a culture of the desired strain.
Incubate the culture ON at 37°C, 230 rpm.
After the incubation ON, add 5 ml of the culture and 5 ml of glycerol 99% into a cryotube.
Conservate the cryotube in a -80°C freezer.

Bacterial induction

Aim
The aim of the bacterial induction is to induce the bacterial production of compounds of interest thanks to the presence of an inducible promoter in the latter. It is used to characterise the strain as well as to produce the compounds of interest to extract and purify them.

Materials

• Bacterial strain

• LB medium

• Antibiotics

• Spectrophotometer

• Induction solvent : IPTG or Arabinose

Procedure

Make a preculture medium by adding LB medium + the antibiotics the strain resists to.
Add 25 µL of a culture of the desired strain.
Incubate the preculture ON at 37°C, 230 rpm.
After the incubation ON, add 25 µL of the preculture into a new LB medium + the antibiotics the strain resists to.
Measure the OD600nm of each sample.
Incubate the culture at 37°C, 230 rpm for 6h.
Measure the OD600nm of each sample.
When he OD600nm of each sample reaches 0.5, add the induction solvent in the samples you wish to induce.
Incubate the culture at 37°C, 230 rpm for the time you wish to make the induction.

SDS page

Aim
The aim of our SDS PAGE is to study the presence of proteins in an induced bacterial sample.

Materials

• Induction samples

• Pre-stain Protein marker

• TEMED

• Ammonium persulfate (APS) 10%

• SDS 10%

• 40% Acrylamide stock

• Isopropanol

• Tris 1.5M (pH 8.8)

• Tris 0.5M (pH 6.8)

• Laemmli 5X

• 1x running buffer

• Protein mini gel cassettes

• Heating block module

• Power supply

Procedure

Sample extraction
- After desired induction time (24,48 or 72h), retrieve 250 µL of the medium for each sample.

- Centrifuge the samples at 10,000 rmp for 3 minutes.

- Discard the supernatants and conserve the pellets at -20°C.

Making SDS-PAGE gel

- Clean and completely dry glass plates, combs, and spacers are required.

- Assemble gel cassette by following manufacturer's instructions.

- Prepare desired percentage of lower gel (separating gel) by adding the following solutions (total volume= 5 ml):

Solutions	One 8% upper gel (1mm)	One 10% upper gel (1mm)	One 12% upper gel (1mm)
H2O	2.65	2.4	2.15
Tris 1.5M pH 8.8	1.25	1.25	1.25
Acrylamide 40%	1	1.25	1.5
SDS 10%	0.05	0.05	0.05
APS 10%	0.05	0.05	0.05
TEMED	0.005	0.005	0.005

- To avoid polymerization, after adding TEMED, mix well and quickly transfer the gel solution by using 1 ml pipette to the casting chamber between the glass plates and fill up to about 0.7 cm below the bottom of comb when the comb is in place.

- Add a small layer of isopropanol to the top of the gel prior to polymerization to straighten the level of the gel.

- Once the gel has polymerized, start to prepare stacking gel (4%) by adding the following solutions (total volume= 5 ml):

Solutions	Two 4% lower gels (1mm)
H2O	3.14
Tris 0.5M pH 6.8	1.25
Acrylamide 40%	0.5
SDS 10%	0.05
APS 10%	0.05
TEMED	0.005

- Remove the isopropanol layer by using filter paper. Rinse the top layer of the gel with ddH2O and dry off as much of the water as possible by using filter paper.

- Add TEMED and mix the stacking gel solution well. Quickly transfer the gel solution by using a 1 ml pipette till the space is full, and then insert the appropriate comb.

- Allow the top portion to solidify and then carefully remove the comb.

Sample preparation
- Add 20µL of H2O + 5µL Laemmli 5X in each pellet of sample.

- Add 15µL of H2O + 5µL Laemmli 5X to 5µL of Prestained protein marker.

- Mix and boil the samples at 95 °C heating block module for 10 min.

- Spin the samples at the maximal speed for 1 min in tabletop centrifuge and leave the samples at room temperature until you are ready to load onto the gel.
Note: Can store extracted protein samples (containing sample buffer) at -20 °C and reheat at 95 °C for 5 min when used the following time.

Electrophoresis
- Remove the gel cassette from the casting stand and place it in the electrode assembly with the short plate on the inside. Press down on the electrode assembly while clamping the frame to secure the electrode assembly and put the clamping frame into the electrophoresis tank.

- For some 1x electrophoresis running buffer into the opening of the casting frame between the gel cassettes. Add enough buffer to fill the wells of the gel. Fill the region outside of the frame with 1x running buffer.

- Slowly load 7µL of protein samples into each well as well as load 7µL of Prestained protein marker.

- Connect the electrophoresis tank to the power supply.

- Run the samples at 150V for an hour.
Protein detection
- Pour Coomassie blue staining on the gels until the gels are covered.

- After 15 min, retrieve the Coomassie blue staining and wash the gels with dH2O

- Pour destaining buffer on the gels until the gels are covered and let it react for une hour or ON.

- Retrieve the destaining buffer and wash the gels with dH2O.

Acidic value

Aim
This test is applied here to quantify the fatty acid content in bacterial culture comparing an induced bacterial culture to a non-induced one. The principle of this test is to measure the volume of potassium hydroxide required to neutralize the free acidity in a sample.

Materials

• Bacterial culture induced and not

• Hexane

• Sodium chloride 10%

• Acetic acid

• Separating funnel

• Phenolphthalein

• Titrator

Procedure

Extract the fatty acids from 50 mL of bacterial culture by adding 6.25 mL of 10% sodium chloride, 6.25 mL of acetic acid and 50 mL of hexane in a separating funnel.
Mix vigorously without forgetting to release the gas.
Let the separating funnel stand without the plug to process the decantation.
Once two separated phases are visible, throw the aqueous phase and collect the upper hexane phase containing the extracted fatty acids.
Add some drops of phenolphthalein to the hexane phase and measure the volume of potassium hydroxide required to turn the solution pink.
The acidic value is then calculated thanks to this formula :

Protein purification with 6-His tag

Aim
To purify the protein of interest with the use of the added 6-His tag.

Materials

• Induced and non-induced samples

• Centrifuge

• Sonicator

• 37°C incubator

• Lysis buffer with protease inhibitor and lysozyme

• Ni NTA beads

• PBS-Tween 20

• Laemli 1X

Procedure

Centrifuge 10 mL of frozen bacterial culture at 5000 rpm for 15 min.
Throw the supernatant and add 200 μL of lysis buffer to the pellet.
Let stand for 10 min at 37°C.
Sonicate 3 times for 20 sec.
Wash the Ni NTA beads 3 times with 500 μL PBS-Tween and the last wash with 250 μL lysis buffer.
Centrifuge the sonicated samples à 15000 rpm for 5 min.
Add 200 μL of supernatant to the cleaned Ni NTA beads.
Let the samples stand for 30 min at room temperature while stirring.
Wash 3 times with PBS-Tween 20 1 ‰.
Add 50 μL of Laemli 1X to the beads pellet.
Centrifuge few seconds and recover the supernatant for SDS-Page.

Enzymatic assay of ADR

Aim
Alcohol dehydrogenase (ADR or ADH) catalyzes the oxidation of alcohol to acetaldehyde with the simultaneous reduction of nicotinamide adenine dinucleotide (NAD) to NADH. The consequent increase in absorbance at 340 nm is directly proportional to alcohol concentration in the sample. This test is applied here to quantify the NADH content in bacterial culture comparing an induced bacterial culture to a non-induced one, in order to deduce the activity of ADR. Ethanol + NAD ➔ Acetaldehyde + NADH

Materials

• Inducted samples

• NAD 10 mM

• Sodium pyrophosphate 100 mM pH 8.5

• Ethanol 1%

• Spectrophotometer

Procedure

Culture O/N bacteria in LB broth with the needed antibiotic, at 37°C and 225 rpm.
Recover 1,5 mL of bacteria in a 1,5 mL Eppendorf tube.
Centrifuge 2 minutes to 9000 rpm to eliminate bacteria.
Transfer the supernant into a new tube.
In another tube, premix 938 μL of sodium pyrophosphate 100 mM, 627 μL of NAD 10 mM and 312 μL of ethanol 1%.
The cell of the spectrophotometer must be heated at 30°C before starting the measurements.
Put 500 μL in 3 spectrophotometer cuvettes. One cell for the blank, another by adding 17 μL of ADR (2.5 units/mL) for the positive control and another with the crude extract.
Follow the production of NADH with OD at 340 nm during 3 minutes. Take the measurement every 20 seconds.

2-nonanone extraction

Aim
The aim of the 2-nonanone extraction is to study its production by a modified bacteria thanks to Gas Chromatography (GC) and High Pressure Liquid Chromatography (HPLC) analysis.

Materials

• Inducted samples

• Decane

• Spectrophotometer

Procedure

After adding the induction solvent to the desired samples, add 1 ml of decane on top of each sample.
Measure the OD600nm of each sample.
Incubate the cultures at 37°C, 230 rpm for the time you wish to make the induction.
After desired time of induction, measure the OD600nm of each sample and retrieve the decane phase of each sample.
If needed, centrifuge the retrieved samples at 3000 rpm for 30 min.
Retrieved the decane phase.
The samples can be directly used with GC and HPLC analysis.

Octanoic and decanoic acids extraction for GC analysis

Aim
The aim of the octanoic and decanoic acids extraction is to study their production by a modified bacteria thanks to Gas Chromatography (GC).

Materials 1

• Induction samples

• Hydrochloric acid

• Ethyl acetate

• Nitrogen gas for evaporation

• Sulfuric acid

• Methanol

Procedure 1

Acidify the medium with 6 M hydrochloric acid to pH < 2.
Extract the acidic materials with equal volume of ethyl acetate.
Evaporate the collected organic layer with nitrogen and then expose the extracts to 3ml sulfuric acid/methanol (1/99, v/v) at 70 °C for 1h to generate fatty acids or HFAs methyl esters (FAMEs or HFAMEs), which can be analysed in GC, unlike free fatty acids.
Extract the FAMEs and HFAMEs with 3ml n-hexane.

Materials 2

• Induction samples

• Sodium Chloride

• Acetic acid

• Ethyl acetate

• Ethanol

• Hydrochloric acid

• ddH2O

• Hexane

Procedure 2

After desired time of induction (24, 48 or 72h), measure the OD600nm of each sample.
Mix 1 mL of induced culture with 125 μL of 10% (wt/v) sodium chloride and 125 μL of acetic acid.
Add 500 μL of ethyl acetate.
Vortex the mixture for 30s and then centrifuge at 16,000×g for 10 min.
Transfer 250 μL of top organic phase to a new glass tube.
Add 2.25 mL of ethanol:hydrochloric acid (30:1v/v) to generate fatty acids or HFAs ethyl esters (FAEEs or HFAEEs), which can be analysed in GC, unlike free fatty acids.
Incubate at 55 °C for 1 h.
Add 1.25 mL of double-distilled water (ddH2O) and 1.25 mL of hexane.
Vortex, and centrifuged at 2000×g for 2 min.
Recover the hexane phase to GC analyses.

4-oxo octanoic and 4-oxo decanoic acids extraction for GC analysis

Aim
The aim of the 4-oxo octanoic and 4-oxo decanoic acids extraction is to study their production by a modified bacteria thanks to Gas Chromatography (GC).

Materials

• Induction samples

• Sodium Chloride

• Acetic acid

• Ethyl acetate

• 5 % 1,2-propyl-enediamine

• Acetic acid/sodium acetate buffer pH3.2

• Water bath

• Chloroform

Procedure

After desired time of induction (24,48 or 72h), measure the OD600nm of each sample
Add 1 mL of 5 % 1,2-propyl-enediamine and 1 mL of acetic acid-sodium acetate buffer pH3.2.
Heat the samples on water bath at 95 ºC for 30 min and allow them to cool at room temperature.
Add 1ml chloroform
Mix well the samples
Let the layers separate
Retrieve the organic layer to make the GC analysis

GC analysis of 2-nonanone

Aim
The aim of the 2-nonanone GC analysis is to study its production by a modified bacteria.

Materials

• Extracted induction samples

• Helium

• HP-5 column (30m, 0.32 cm inner diameter, 0.25µm particles diameter)(Agilent)

Procedure

Each sample is injected at 1µL in the apparatus.
The apparatus has a split of ⅕.
The injector temperature is set at 250°C.
The oven temperature is set at 40°C from the beginning to 1min.
The oven temperature is set from 40°C to 150°C with an increase of 37°C/min.
The oven temperature is set from 150°C to 300°C with an increase of 100°C/min.
The oven temperature is set at 300°C for 3 min.

GC analysis of octanoic and decanoic acids

Aim
The aim of the octanoic and decanoic acids GC analysis is to study their production by a modified bacteria.

Materials

• Extracted induction samples

• Helium or nitrogen

• HP-5 (30m, 0.32 cm inner diameter, 0.25µm particles diameter)(Agilent) or BPX-70 (30m, 0.22 cm inner diameter, 0.25µm particles diameter)(SGE Analytical Science) columns

Procedure

Each sample is injected at 1µL in the apparatus.
The apparatus has a split of 1/5.
The injector temperature is set at 250°C.
The oven temperature is set at 50°C from the beginning to 2 min.
The oven temperature is set from 50°C to 200°C with an increase of 25°C/min.
Hold the temperature for 1 min.
The oven temperature is set from 200°C to 315°C with an increase of 25°C/min.
The oven temperature is set at 300°C for 2 min.
The results are available on the GC software on the computer as a curve with the time of the analysis as the X-axis and the pico amperes (pA)or microvolts (µV)(depending on the GC aparatus used), which is the amplitude of detection of a compound detected by the Flame Ionization Detector (FID) as the Y-axis.

GC analysis of 4-oxo octanoic and 4-oxo decanoic acids

Aim
The aim of the 4-oxo octanoic and 4-oxo decanoic acids GC analysis is to study their production by a modified bacteria.

Materials

• Extracted induction samples

• Helium

• HP-5 column(30m, 0.32 cm inner diameter, 0.25µm particles diameter)(Agilent)

Procedure

Each sample is injected at 1µL in the apparatus.
The apparatus has a split of 1/5.
The injector temperature is set at 250°C.
The oven temperature is set at 180°C from the beginning to 2 min.
The oven temperature is set from 180°C to 230°C with an increase of 10°C/min.
The results are available on the GC software on the computer as a curve with the time of the analysis as the X-axis and the pico ampere (pA) which is the amplitude of detection of a compound detected by the Flame Ionization Detector (FID) as the Y-axis.

Octanoic, decanoic, 4-oxo octanoic and 4-oxo decanoic acids extraction for HPLC analysis

Aim
The aim of the Octanoic, decanoic, 4-oxo octanoic and 4-oxo decanoic acids extraction is to study their production by a modified bacteria thanks to High Pressure Liquid Chromatography (HPLC) analysis.

Materials

• Induction samples

• Decane

Procedure

After desired time of induction (24, 48 or 72h), measure the OD600nm of each sample.
Mix 6 mL of induced culture with 1ml of decane.
Vortex the mixture for 1min and then centrifuge at 4,000×g for 15 min.
Transfer the top organic phase to a new glass tube.
Repeat the steps 2 to 4.
Evaporate at 60 °C in nitrogen until it is dry.
Add 300µl of Acetonitrile to retrieve the sample.
Samples can directly be used for HPLC analysis.

HPLC analysis of 2-Nonanone, octanoic and decanoic acids, 4-oxo octanoic and 4-oxo decanoic acids

Aim
The aim of the HPLC is to identify compounds from a solution. It can also be used to purify them, when parameters as retention time and eluent concentration are known.
As the HPLC characteristics of our molecules are unknown, we will also use chemical standards to create a data bank of these characteristics, to be able to determine if our future strains produce our molecules of interest.

Materials

• IC8, C10, 2-nonanone, 4OOA and 4ODA Compounds’ standards

• Vacuum system

• Sonication bath

• Magnetic stirring

• Stationary phase - HPLC column: C18 column Luna Omega LC column from phenomenex

• Mobile phase :

- Acetonitrile 100% HPLC Grade

- Water 100% HPLC Grade

Procedure

Standards are prepared at the wanted concentrations in acetonitrile 100%. In our case, each standard is prepared at 10 g/L and 100 g/L.
Prepare the solutions: solution A is 30% acetonitrile-70% water, solution B is 100% acetonitrile. Both of them contain 0.2% Acetic Acid.
Degas solutions. The water-acetonitrile needs almost 1h30-2h of degassing, acetonitrile needs around 1h-1h30. To do so, bottles are plugged in the vacuum system, one bottle in sonication bath, the other bottle with magnetic stirring.
Parameter the analysis HPLC program:
- Hold 100% of the solution A for 10 min

- Go from 100% to 0% A over 7 min (at the same time, program 0%-100% B)

- Hold 100%B for 8 min

- Come back to 100% A in 5 min
A run without sample is recommended, but not obligatory
Inject your sample and start the program

With this program, the acetonitrile concentration passing through the column will vary between 30% and 100% as represented below. The machine adjust the ratio between the two solutions automatically.

Fig 1: Acetonitrile concentration variation during the HPLC program.

Team:Poitiers/Protocols

Protocols