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Spherical silica and irregular silica: what differences?


Silica gels structure

Silicas, used in liquid chromatography, are divided into two main categories:

  • Silicas with irregular particles
  • Silicas whose particles are spherical

If the first ones are still widely used In the field of flash chromatography or industrial scale purification, the latter are now the absolute standard for (U) HPLC analysis.

But what are their advantages or disadvantages?
Could we indifferently invert them in their respective use?

To answer, let’s first examine the structure of silica. Chemically it consists of silicon atoms and oxygen. Each silicon atom is mobile and linked to oxygen atoms. The general structure can be represented as well :

General structure of silica

 

Silica gels shapes

Irregular or spherical, the most common silicas retain this chemical structure. When they are synthesized, the processes implemented lead to the creation of particles of the forms that interest us. Spherical silicas resemble small porous beads whereas irregular particles are similar to small pebbles that are also porous.

Silica gels shapes

 

Physico-chemical characteristics

These silicas are characterized by chemical and physical values that give them special properties essential for separating chemical compounds with very varied structures.

Physical

• Geometry (irrégular, spherical)
• Particle size (dp in µm)
• Pore diameter (Angström)
• Pore volume (ml/g)
• Specific surface area (m2/g)
  

Chemical

• Nature and type of bonding
• % Coverage (carbon %, coverage rate/m2)
• Type of silica (pure or not)

 

Stacking of silica gel in a column

A Flash, (U) HPLC or preparative column filled with these silicas will be more or less effective depending on the particle size of the media. It is best estimated that a plate (separation stage) can not be smaller than the diameter of the silica particle. As a result, the smaller the particle, the more “plates” in the column. In the example below, whatever the theoretical model, it is deduced that particles of 15μm will have a stacking 2 times more compact than particles of 30μm.

The irregular silicas show, as their name indicates, indefinite shapes for which it is difficult to measure the mean diameter. In addition, their stacking is disordered and resulted in a much lower compactness than the spherical silicas. Finally, these silicas usually contain many “fines”, that are, small fractions that can pass through the columns frits.

The arrangement of spherical silicas is much greater than irregular silicas. Solvent flow through the column follows a more linear path. The analytes families are less dispersed and exit the column in a smaller solvent volume. Peaks are finer and more Gaussian.

Stacking of silica

Conclusion

It is therefore easy to understand the interest of spherical silicas for who are seeking a more efficient separation. Silicas with smaller particle sizes offer the possibility of reducing the lengths of the columns and therefore the elution time while maintaining a good separation.

For modern analysis and quantification, the use of spherical silicas is obvious. For purification, the best compromise must be found between the price of the adsorbent and the separation efficiency.

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Peptides purification development in Reverse Phase


Introduction

Reverse phase chromatography is a technique widely used to purify peptides. This technique owes its popularity to the fact that the purification times are quite short and the efficiencies achieved. The mobile phases used (mainly acetonitrile and water) are volatile which facilitates the concentration / lyophilization steps.

 

Sample preparation – peptides’s solubility

The solubility of the peptides in solution depends on several parameters:

  • The amino acid sequence (if hydrophobic residues are present, this reduces the solubility in an aqueous medium)
  • The size and the tertiary structure of the peptides. The solubilization can change depending on the distribution of the residues
  • Percentage of residues loaded (it will be necessary to apply particular pH to help solubilization).

There are some rules to guide in the solubilization in aqueous medium:

  • Peptides consisting of less than 5 residues are generally soluble unless the residues are hydrophobic (tryptophan, isoleucine, leucine, phenylalanine, methionine, valine or alanine).
  • Hydrophilic peptides containing more than 25% of charged residues (glutamic acid, asparagic acid, lysine, arginine and histidine) and less than 25% of hydrophobic residues are generally dissolved in an aqueous medium, provided that the charged residues are distributed in the whole sequence
  • Hydrophobic peptides containing from 50% to 75% of hydrophobic residues may be insoluble or partially soluble in aqueous solutions, even if they contain 25% of charged residues (generally, they are soluble in TFA and formic acid
  • Acid peptides (residues Glutamic acid and Asparagic acid) and basic (residues Ariginine, Lysine, Histidine) are more soluble at neutral pH than acidic pH.
  • The highly hydrophobic peptides that have more than 75% hydrophobic residues do not dissolve in aqueous solutions, it is preferable to make a solid deposit to properly inject these peptides.

Peptide sequences comprising a very large proportion of Serine, Threonine, Glutamic acid, Asparagic acid, Lysine, Arginine, Histidine, Asparagine, Glutamine or Tyrosine may form an intermolecular hydrogen bond network and have a tendency to form gels in aqueous solution. When they are concentrated, these peptides can be treated as hydrophobic peptides.

Of course, before passing the sample on the column, it is preferable to test the solubility of a small part under the method start conditions. It’s better to choose a solvent easily removable by lyophilization to recover the solutes as pure as possible.

 

How are the peptides eluted?

The interactions that manage reverse-phase peptide purifications are between the stationary phase and the hydrophobic scaffold of the peptide. They are non-dispersive and low-selective with 2 main mechanisms that are adsorption and sharing.

Small peptides (with molecular weight less than 3kDa) are eluted by a sharing mechanism while the larger peptides are eluted mainly by an adsorption mechanism. That is to say that the molecules at the injection “sticks” against the stationary phase. Then they are eluted when the proportion of organic solvent reaches a defined percentage.

Retention according to the volume fraction of acetonitrileRetention according to the volume fraction of acetonitrile

 

Choice of the stationary phase

Choice of the stationary phaseThe choice of the stationary phase depends on the length of the peptide chains of the solutes which can limit the interactions by steric genes.

A C18 column will preferably be used for peptides of small and medium size, followed by a larger C8 peptide and C4 for polypeptides.

The second parameter to take into account is the porosity of the phase to be used. In fact the longer the peptide will be, the harder it will be to pass it into a small pore. Peptides from 1 to 5kDa will separate with a pore size of 100A, those of size between 5 and 20 kDa can be separated with a pore size of 200A and the polypeptides will be used with a pore size of 300A.

The last point affects the size of the particles. Given the difficulty of some purifications, it is not interesting to use sizes greater than 30µm.

 

Choice of solvent conditions

The most used solvents are water and acetonitrile. Acetonitrile allows to increase the elution of peptides. The fact that this solvent does not respond in UV and is easily removable, makes it an obvious choice.

It is common to use at the same time trifluoroacetic acid (TFA) as an ion-pairing agent which makes it possible to reduce the ionic interactions between the peptides and the stationary phase. Moreover, the high volatility of the TFA makes it a good element as easily removable (be careful, TFA must be present in the same proportion in the two solvents). The disadvantage of this product is that it will make the detection of MS difficult. It is then necessary to lower its concentration but it degrades the profile. As an alternative, you can use another agent such as formic acid that allows detection.

For applications with very hydrophobic compounds it is possible to use isopropanol. This solvent with a greater eluent power has another advantage, not negligible, keeping the biological activated of the peptides. This solvent is mainly used for polypeptides on C4 300Å column.

Generally we start the method with 5% of strong solvent, to increase the eluent force of 1% / min, which allows to have a better resolution.

For applications with polar peptides, use a HILIC mode.

 

Influence of some parameters

Several parameters are important to optimize the purifications:

  • Gradient: As explained in the mechanism part, the peptides (> 3kDa) are eluted by an adsorption mechanism. A step gradient for elution may be useful.
  • ion matching reagent. TFA is the most used but it is possible to use FA, PFPA, HFBA (anionic agent) as well as TEA, TBA (cationic agent) which can then help the separation of peptides with basic residues.
  • Temperature: the temperature of the column is an optimization parameter. It reduces the viscosity of the solvent (allowing a significant reduction in back pressure) while allowing to reduce the time of release of the products. Be careful, however, not to degrade the solutes with too high temperature.
  • Flow: If for the small molecules the flow is an optimization parameter allowing to save time with a small loss of resolution, for the more important peptides it is quite the opposite : an increase of the flow will cause a degradation of the profile of purification.

Van Deemter curveFor peptides of low MW, there is an optimal flow (Van Deemter curve ….)
For peptides of high MW, the rise in flow alters the profile of the peaks. It can even cause peak duplication if the flow rate is too high.
Tyrosine: 181 g/mol
Myoglobin: 17 000g/mol
Albumin: >60 000g/mol

 

If no retentionRetention too high
or no elution
Improve selectivity
Check the solvent of the sample (the eluting force must be less than or equal to the eluting force of the mobile start phase of run)

Check the concentration of the ion-pair reagent (usually the reagent is volatile)
Check the packing of the column (avoid leaving the column under more than 95% organic solvent)

Decrease the concentration of organic solvent

Decrease gradient slope
Check the nature and concentration of the matching reagent

Increase organic solvent concentration

Increase temperature

Change column (test column C8 or C4 if C18 does not work)
Modify the ion pairing reagent

Modify gradient slope

Modify the organic solvent (for example mixture Acetonitrile / IPA, 90/10 max, replacing Acetonitrile alone for very hydrophobic peptide)

Changing column (C18, phenyl, C8, C4)

 

Find out more :

 



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All you need to know about Interchim® pre-packed prep-LC & DAC columns


1. Interchim® pre-packed prep-LC columns

Interchim® Preparative columns range from 10.0 to 50mm i.d for the purification of samples ranging from mg to g.

 

# Column hardware  & column packing

Interchim Dry-load

The tube polishing value (Ra) has a fundamental importance in preparative chromatography.
A primary reason for peak broadening and low efficiency is the use of a poorer hardware quality.
As the mobile phase is slowed down near the column wall, molecules in the center of the mobile phase stream move faster than the molecules closer to the side.
All columns have extremely smooth internal surfaces (typically 8 μ inch of Ra) to considerably reduce issues of drag and maintain column efficiency. Efficiency is also managed through Interchim®’s state-of-the art proprietary packing processes – Modulo-cart Prep withstand packing pressures up to 550 bars contributing strongly to a good bed stability and column life time.

 

# Sample dispersion

The loading of sample onto a preparative column requires stringent management to establish quality separations. Column overloading results in a poor retention of pure fraction and therefore particular attention needs to be placed upon selecting the appropriate column dimension and the properties of the stationary phase. In addition, a careful control of the introduction of sample to the column is necessary to establish a homogeneous sample dispersion through the sorbent bead head. Sample typically enters a preparative column through a 1/16” fitting; poor sample loading will lead to overloading certain areas of the stationary phase whilst other areas will be underloaded.
E.g. For a 50mm i.d column with a 500μm i.d capillary fitting – sample introduced onto the column (without any sample distributor) will only interact with 0.01% of the surface column head. As well as a dramatic loss in capacity there will also be a high potential for the column head to prematurely clog, rapidly reducing column life times.
To prevent this problem Interchim®’s Modulo-cart Preparative columns are outfitted with a sample distributor. The sample distributor design maximizes the efficiency of sample volume dispersion and the sample mass introduced to the surface of the column head raising column life time.

 

 

2. Interchim® DAC columns

Interchim DAC ColumnsDAC stands for dynamic axial compression. It combines the preparative column and packing system together. It is very simple to operate. The column can be used online when it is packed. Don’t take the column down!
The piston of the column always produces a stable pressure on packing bed which prevent the collapse and loose of the column bed.
They can be packed with small particulate media to reach high levels of performance.

  • Column tube material: 316L
  • Roughness: Inner surface Ra ≤ 0.4µm
  • Filter:  316L Pore size 3-5µm
  • High pressure seal PTFE and 316L
  • Operating temperature: 5-60°C
  • Control pannel: air pressure gauge, oil gauge, regulating valve, emmergency stop switch, change direction valve, shutt-off valve
  • Air source: ≥ 6bar, output ≥ 8m3/min
P/NFormatIDMax bed heightInlet/Outlet connectionOverall dimensionsWeight
KV7350DAC ID5050mm300mm1/16″550mm x 500mm x 1900mm100kg
KV7370DAC ID8080mm300mm1/8″550mm x 600mm x 2200mm200kg
KV7390DAC ID100100mm300mm1/8″550mm x 600mm x 2200mm200kg

 

Learn more:

Feel free to contact the technical support at +33 4 70 03 73 01 or by email at interfine@interchim.com.



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Temperature Effect on PhotoRedOx Chemistry


Temperature control of photoredox experiments is not easily accomplished. Most experiments are described using a fan to maintain the reaction near room temperature and no fan to let the reaction warm up. Moreover there are very few setups described allowing reaction to be performed below room temperature. In this context it is very challenging to study temperature effect on photocatalysis in a convenient way.

 

PhotoRedOx TCHepatoChem developed a device PhotoRedOx TC that makes this type of experiments easy to perform using a simple chiller/heater recirculator.


The PhotoRedOx TC (for Temperature controlled) fits many light sources like for example the LEDs EvoluChem 18W. It also has a photochemistry chamber to evenly distribute light. Regarding the format vials, this apparatus is very flexible (from 0.3ml to 20ml) thanks to the availibility of many racks. Flow reactor can also be used with this device. PhotoRedOx TC required to use a magnetic stirring plate to provide agitation and an external fluid circulator to heat or cool the reaction vessel. (light sources, racks, flow reactor, stirring plate and fluid circulator are supplied independently)

In the following examples, HepatoChem shows how CH alkylation using a BF3K reagent can benefit from lowering temperature of the reaction and how increasing the temperature can improve conversion of a C-O cross-coupling.

 

PhotoRedOx TC (Temperature Controlled)

  • Fits many light sources (EvoluChem 18W)
  • Photochemistry chamber to evenly distribute light
  • Flexible format vials (from 0.3mL to 20mL)
  • Flow reactor available
  • Stirring on magnetic stirring plate
  • External recirculatorneeded to heat or chill reaction vessel

 

BF3K Cyclopropyl and hydroquinine reaction

Reaction_Cyclopropyl BF3K & Hydroquinine

Reaction Conversion at 19°C, 28°C and 50°C

Reaction Conversion at 19°C, 28°C and 50°C

Experimental Details: Reaction performed in Evoluchem Photoredox Temperature control bath with circulating polyethylene glycol/water bath and an EvoluChem 18W 6200K white light for 2 hr. Reaction contains 50 µmol substrate, 1.5 equiv. RBF3K, 2 equiv. K2S2O8, 5 equiv. TFA and 2 mol% Ir(dF-CF3-ppy)2(dtbpy) in 0.5 ml DMSO.

 

C-O Coupling with Cyclohexanol

Reaction Conversion at 33°C and 50°C

Reaction Conversion at 33°C and 50°C

C-O Coupling with Cyclohexanol

Experimental Details: Reaction performed in Evoluchem Photoredox TC box with EC 450 nm LED. Reaction 2 mol% Ir(dF-CF3-ppy)2(dtbpy), 5 mol% NiCl2-dme/dtbpy and 3 equiv. base with 10 mol% quinuclidine. Conversion determined by LC-UV.

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Solutions for low temperature synthesis


Some chemical syntheses must be done at low temperatures over long periods of time.

To have the possibilities to do that, several solutions can be considered, like the use of:

A mixture of ice – salt:

Minimum temperatures of a ice-salt mixtures:

  • T ~ 0°C: ice bath.
  • T ~ -10°C: ice bath (70 %) + calcium chloride (30 %).
  • T ~ – 15 °C: ice bath (80 %) + amonnium chloride (20 %).
  • T ~ – 20°C: ice bath (75 %) + sodium chloride (25 %).

Percentages are given in mass.

 

An organic solvent mixture – dry ice:

Mixtures can be obtained bya  mixing of dry ice and organic solvents.

  • T ~ – 23°C: carbon tetrachloride / dry ice.
  • T ~ – 60°C: chloroform / dry ice.
  • T ~ – 78°C: acetone/ dry ice.
  • T ~ – 95°C: toluene/ dry ice.
  • T ~ – 100°C : ether/ dry ice.

 

The air or a liquid nitrogen or an organic solvent / liquid nitrogen:

  • T ~ – 170 – 180°C

 

Precautions to be taken and constraints encountered:

For each solution, some cautions should also be considered to maintain low temperatures, such as:

  • Add a thermal insulation of the synthesis system by covering it with an insulating material.
  • Use of a glass Dewar or Cool-It.

Despite these solutions and cautions, the stability of the temperature of the reaction medium will not be guaranteed and becomes very annoying for the synthesis of boronic acids.

Boronic acids can be done creating a magnesium or a lithian and then add it on a boric acid ester (Scheme 1) and then hydrolyze the arylboronic ester obtained and then recover the boronic acid.

 

Addition arylmetallique sur trialkylborateBoronic acids done from the organolithians have the advantage of preparing the aryllithians by ortholithization, freeing themselves from the halogenated precursor. It has also been shown that in some cases the unstable organolithien can be trapped in situ by adding the lithic base to a mixture of arene and borate (Scheme 2).

Borylation by ortholithiation & entrapment in situ

Limitations for the ortholithiation is that this synthesis must be done at very low temperatures (from -78°C to -40°C) with often, limited existing synthesis tools with which the need for monitoring the temperature due to frequent recharge of carboglace/solvent or liquid nitrogen/solvent, by the users, can quickly become cumbersome and time-consuming.

 

Push the limits with efficients solutions:

In order to push these limitations Interchim can support you offering effective and appropriate systems for specific applications.

For example, for applications where the stability of the temperature and an efficient stirring are required over long period time Interchim recommands:

For synthesis done in 10ml up to 2L round bottom flasks:

• The use of the Cool-It bowl from Radleys and the immersion cooler TC100E from Huber.

Reservoir incassable Cool-itThe unbreakable HDPE Cool-it
insulated bowls fit onto standard
stirring hotplate for round bottom flasks.
Cryoplongeur TC100EThe immersion cooler TC100E
allows a quick and a controlled cooling
of the liquid inside the Cool-It bowl
and to garantee a constant temperature
(T°C max: -100°C) with an accuracy of +/- 0.5k.
The combination of these two systems ensures the stability
of temperature of the reaction medium and an effective stirring
over an unlimited reaction time.

 

For synthesis done with parallel reactions stations:

• The use of the Cooled Carousel Reservoirs from Radleys and the immersion cooler TC100E from Huber.

Reservoir incassable Cool CarouselThe unbreakable HDPE Cooled
Carousel Reservoirs fit onto standard
stirring hotplate and can be used
with 6 or 12 or 24 parallel reaction stations.
Cryoplongeur TC100E avec Cool CarouselThe immersion cooler TC100E
allows a quick and a controlled cooling
of the liquid inside the Cooled Carousel Reservoirs
and to garantee a constant temperature
(T°C max: -100°C) with an accuracy of +/- 0.5k.
The combination of these two systems ensures the stability
of temperature of the medium and an effective stirring
over an unlimited reaction time.

 

For more information:

In all of this, to help you push back the limits of your syntheses because of the existing tools, contact Interchim®.

Thanks to our big network of suppliers everywhere in the world, Interchim will be always able to find the best solution with innovative productivity tools to answer exactly to the needs requested by your applications.

Caroussel_6_Plus_interchim_blog1218Caroussel 12 PlusHuber_Temperature_Control_interchim_blog1218

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Feel free to contact the technical support at +33 4 70 03 73 01 or by email at interfine@interchim.com.



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Lead Diversification Tool Box from Hepatochem


Lead Diversification tool box

Illustration_interchim_blog0618

HepatoChem has developed several kits for lead diversification for the C-H functionalization. This chemistry offers many possible transformations including hydroxylation, acetoxylation, methoxylation, and halogenation among others. This chemistry can be sometimes difficult.
The HepatoChem kits is offering you a rapid, practical and cost-effective solution for analogues obtaining. They enable the parallel screening of a selected set of catalytic conditions focusing on generating diversity with an approach totally orthogonal and complementary to conventional synthetic methods.

HepatoChem proposes different kits:

  • Alkoxylation and Acetoxylation kits
  • Fluorination kit
  • Biomimetic Oxidation kit
  • Sulfinate Alkylation reagent kit
  • Photocatalytic alkylation diversification kit

 

Alkoxylation and Acetoxylation Kits

Diversity Kit : 4 different functionalizations

C-H Alkoxylation is one of the most C-H functionalization described in literature. HepatoChem kit is designed to enable conveniently the screen of both Alkoxylation and Acetoxylation reaction conditions. It uses PdOAc2 as catalyst with several oxidants and additives.

Kit HCK1007-01-001 (article 9H4350)
Substrate solution is prepared in DMF (to facilitate the screening of solvents) and added to all four for solvents. Screen the four solvents (methanol, ethanol, isopropanol and acetic acid) with three reaction conditions (different salts). 10mol% Pd(OAc)2, 1 equiv PhI(OAc)2.

TableauKitHCK1007-01-001_interchim_blog0618

 

Salt Effect on C-H functionalization

Optimisation Kit: 5 differents salts

It has been reported that salt can influence C-H functionalization. HepatoChem kit is designed to enable the screen of several salts simultaneously. This kit contains 5 different salts CuOAC2, Ag2CO3, K2CO3, Cs2CO3, MgSO4.

Kit HCK1007-01-002 (article 9H4360)
Screen 1 solvent with 12 reaction conditions. 10 mol% PdOAc2, 1 or 2 equivalents of PhIOAc2 with 5 differents salts. Prepare one solution in solvent or mixture in DMF if solubility issues.
Tableau Kit HCK1007-01-002

 

Fluorination Kit

Fluorination is one of the most interesting C-H functionalization described in literature. HepatoChem kit is designed to screen fluorination reaction conditions using PdOAc2  as catalyst in presence of common fluorine sources : Silver Fluoride AgF, 1-fluoro-2,4,6-trimethyl-pyridinium triflate (TMPyF), SelectFluor®, N-fluorobenzenesulfonimide (NFSI) and Bis(tert-butylcarbonyloxy iodobenzene (PhIOPiv).

Kit HCK1008-01-001 (article APQ9X0)
This kit includes 2 sets of reagents ; catalyst and oxidant are mixed in the same vial

12 conditions with AgF, TMPyF, Selectfluor and NFSI

Kit HCK1008-01-001

 

Biomimetic Oxidation Kits

HepatoChem has developed a revolutionary way to screen, optimize, and produce metabolites directly from drug candidates. The BMO kit exploits an optimized panel of catalytic chemical reaction conditions with organometallic catalysts. This tool enables to mimic the suite of cytochrome P450 enzymes (CYP) present in human hepatocytes.
HepatoChem BMO kits enable, in three simple steps, synthesis of metabolites directly from the parent drug.

Biomimetic Oxidation Kits

BMO Screening kit : HCK1001-01-001 (article 9H4040)

Perform the primary screen, select the desired metabolites wells. Then order the corresponding optimization kit.
The complete kit contains all solvents and reagents for 2×25 screening reaction conditions (2 plates included).

BMO Optimization kit : HCK1001-02-XXX (article 9H4050)
Perform the optimization kit, identify the best production conditions and then order the corresponding production kit.
The complete kit contains solvents and reagents for the optimization of selected screening reaction conditions (1 plate included).

BMO Production kits : HCK1001-03-XXX-02 (article 9H4060)
and HCK1001-03-XXX-10 (article 9H4070)

You can scale-up and produce your metabolite
The complete kit includes all solvents and reagents for your metabolite at 12.5µmol scale or more.

 

Sulfinate Alkylation Reagent Kit

The sulfinate alkylation reaction described by Prof. Baran is a powerful late stage functionalization tool. HepatoChem kit enables to produce analogues of the lead compound in mg quantities using 6 different alkylation reagents. Each vials contains 100µmol of sulfinate alkylation reagent and a stirring bar to react with 50µmol of substrate. The C-H functionalization will mainly occur on electron-deficient heteroarenes at one or several positions.

Kit HCK1013-01-001 (article AYHDQ0)
kit content: 2 reaction vials of each reagents (100µmol), 12 reaction vials total

Sulfinate Alkylation Reagent Kit


Tableau Sulfinate Alkylation Reagent Kit

 

Photocatalytic Alkylation Diversification Kit

A photoredox kit is also available for the C-H functionalization

The trifluoroborate alkylation reaction (Minisci reaction) described by Prof. Molander is a powerful late stage functionalization tool. HepatoChem kit enables to produce in one step 8 different analogues of a lead compound in mg quantities. C-H functionalization will mainly occur on electron-deficient heteroarenes at one or several positions.

Kit HCK1016-01-001 (article AYQRA0)
Kit contents (HCK1016-01-001) : 2 reaction vials of each BF3K reagents (75µmol) and K2S2O8 (100µmol), 2 vials of photocatalysts and 2 vials of TFA, 16 reaction vials total

KitHCK1016-01-001_interchim_blog0618

Tableau KitHCK1016-01-001



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Let the magic happen : Merry christmas from France


The end of the year brings us no greater joy than the opportunity to wish you a Merry Christmas ! May it be a wonderful season for you and yours. Thank you for being part of this magic throughout the year. 🙂



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puriFlash monolith columns


The purification of compounds is always a compromise between desired purity, the sample load and the duration of methods. To improve efficiency in obtaining pure compounds, chemists must find the best balance between purity, duration of the method, and environmental considerations. This delicate balance is often necessary for both raw products and final purification.
Due to their particular structure, the puriFLash® Monolith columns provide a great help for all the points on which a balance must be implemented.

What is a puriFlash® monolith column?

Interchim® Peptides monolith column is a pre-packed column with the novel silica gel for reversed-phase liquid chromatography that will permit high-speed processing only with a medium to low back pressure.

Microsoft PowerPoint - BioProcess Poster v3 Final.pptx

How puriFlash®monolith columns can provide best results?

In the pores of this monolith, there are two structures (Macro and micro pores), which allow a faster and deeper diffusion of the solvent inside the particles. That conduces to a more effective purification, especially of macromolecules such as peptides, with an extremely low pressure.

Particle of 30µm which provide equivalent result of 15µm and less pressure

When used with the optimum flow rates, these 30μm phase-fill columns will give you at least 15μm phase results, as shown in the comparison below.

ColonnespuriFlashMonolith2_interchim_blog0518

Ultra-High throughput

Increase the flow-rate of purifications to the limit pressure of your columns and significantly reduce the time of purifications.
As these columns generate less pressure, it becomes possible to use flow rates higher than the optimum flow of your columns, without losing the separation’s quality for better productivity. This example shows that it is possible to use 10x the optimum flow without losing the quality of separation. This product therefore allows better productivity.

ColonnespuriFlashMonolith3_interchim_blog0518

This advantage can even be used when a purification requires to stack columns for difficult purifications. Then, it will be possible not only to stack columns, but also to work at a higher flow rate than the optimum, which is not possible with conventional silica.

Example :
Sample:
1- GLY-TYR 238
2- VAL-TYR-VAL 380
3- Met-Enkephalin 574
4- Angiotensin 1 000
5- Cytochrome c from bovine heart 11749

Method :
Solvant A : Water + 0.1%TFA
Solvent B : ACN + 0.1%TFA

ColonnespuriFlashMonolith4_interchim_blog0518

Gradient : 5 to 40% d’ACN in 33 :45
Débit : 15 mL/min
Pression : 4 bar (monolith)
7 bar (PF-15C18N-F0025)

For this application, the two columns give a similar result.
The compounds 3 and 4 are co-eluted.

 

ColonnespuriFlashMonolith5_interchim_blog0518

Gradient : 5 to 40% d’ACN in 66 :30
Débit : 15 mL/min
Pression : 4 bar (monolith)
13 bar (PF-15C18N-F0025)

With the stack of two columns we get to separate compounds 3 and 4 but the run time is then doubled exceeding the hour!

 

ColonnespuriFlashMonolith6_interchim_blog0518

Gradient : 5 to 40% d’ACN in 16 :30
Débit : 60 mL/min
Pression : 17 bar (Monolith)
Impossible avec PF-15C18N-F0025

When increasing the flow-rate, even slightly, the column PF-15C18N-F0025 generates too much pressure, so it is not possible to significantly reduce the run time.
On the contrary, the monolith column makes it possible here to increase the flow rate to 60mL / min (4 times the optimum flow rate) and makes the purification time to 16min.
The time saving is important.

Less toxicity

Thanks to the pressure, it’s possible to use viscous solvent as isopropanol. Free from toxics solvents as acetonitrile and methanol.
In this application, eluent solvent is IPA/Water, 1:1, we see that the generated pressure is under max pressure of F0025 column.

ColonnespuriFlashMonolith7_interchim_blog0518

 

Enhanced Performance with Any System!

Even in the reverse phase purification, the pressure of Interchim® puriFlash® monolith column is as low as 2 bar or less, at a standard flow rate and can be adapted to any low / medium pressure machine like puriFlash® machines. Moreover, it is quite easy to improve separation performance by stacking 2 or more columns.

Why to choose puriFlash®monolith columns?

As the different results show, the use of puriFlash® monolith columns will help you to:
  – save up to 80% of run-time on your purifications and increase your productivity.
  – use columns with a particle size of 30 μm and obtain results at least equivalent
     to 15 μm columns.
  – get rid of toxic solvents such as acetonitrile and methanol, and even use solvents
     generating high back pressure such as isopropanol.
  – use small particle size columns on low pressure systems for better performance.

 

Learn more :

Find the complete list of columns here.



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Analytica 2018, here we go !


Analytica 2018 is coming ! We look forward to welcoming you to our booth ! 🙂

See you in Germany or follow us with our social medias!

Analytica 2018 - Interchim



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Bring Your Chemistry to Light with the PhotoRedOx Box


PhotoRedOx Setup

Interest in photochemistry has been growing exponentially in recent years. Numerous new applications using visible-light photoredox catalysis have been discovered. These catalytic systems can perform many types of bond formations using various substrates which are valuable new tools for synthetic chemists.
However photoredox chemistry setup necessitates to the use of a light source (blue light) and apparatus that are not standard yet in an organic chemistry laboratory. Many chemists have made their own setup and tried to reproduce literature chemistry with more or less success. As a result the implementation of photoredox chemistry is slow and organic chemists are still hesitant to try these important new tools. Therefore, the need for a simple and robust device to perform visible-light photoredox catalysis has become increasingly important.

EvoluChem™ PhotoRedOx Box

The EvoluChem™ PhotoRedOx Box was designed with one main objective: To allow any chemist to easily perform multiple photoredox reactions in a reproducible environment. Our photochemistry device provide an even light distribution to all reaction samples allowing consistent and reproducible reactions. A cooling fan allows even temperature distribution and keeps the chamber near room temperature during long reaction runs. The device easily fits on standard stir plates, allowing for consistent stirring. Sample holders are compatible with vials ranging from 0.3 ml to 20 ml vials.

Unique Design

Schema PhotoRedOxThe PhotoRedOx Box is using a unique geometry of mirrors to irradiate multiple samples simulatanously for parallel chemistry setup while limiting the thermal effect of the light source. This design results into a compact and efficient photoredox device which can be easely set on any standard stir plate.
The removable lamp adapter allows easy switching from the standard kessil™ blue 34W LED lamp to many other light sources.

Fit multiple vial formats

PhotoRedOx_Box_interchim_blog0218Organic chemists needs to be able to use different reaction vial sizes depending on the scale and the number of the reaction to be performed. The PhotoRedOx Box can virtually fit any type of vials including 0.3ml crimped vials (6 x 32mm), 2ml HPLC vials (12 x 32mm), 1DRAM (15 x 45mm), Microwave vial 2-5mL (17 x 83mm), 2DRAM (17 x 60mm) and 20ml scintillation vials (28 x 61mm).

 

This feature allows quick and consistent scale up from screen reactions to larger scale with preset sample positions removing the guess work on sample placement distance from the light source. When using 0.3 ml vials, 32 reactions can be performed in parallel in the photochemical device. At 20 ml, two reactions can be run in duplicate.

Available Holders

PhotoPortoirsRedOx_Box_HCK1006-01-017_interchim_blog0218PhotoPortoirsRedOx_Box_HCK1006-01-018_interchim_blog0218PhotoPortoirsRedOx_Box_HCK1006-01-019_interchim_blog0218PhotoPortoirsRedOx_Box_HCK1006-01-020_interchim_blog0218PhotoPortoirsRedOx_Box_HCK1006-01-021_interchim_blog0218

Reproducibility

With the EvoluChem photomethylation kit, we have demonstrated the reproducibility of both the photomethylation kit and the device. Using a photomethylation of buspirone as test reaction, 16 vials spread through the 0.3 ml vial sample holder for Trial #1 results in 53% (+/-2 %) conversion. See figure. For a second trial with 16 reaction vials we observed an average conversion of 56% (+/-2 %) for the mono-methylated product.

Test rection (Methylation)

Pourcentage de produit de mono-méthylation par position des flacons réactionnels

Reaction conditions:

Each reaction vial contains Ir(dF-CF3-ppy)2(dtbpy)[PF6] (0.1 μmol), tert-butylperacetate solution (12.5 μmol) and a stir bar sealed under inert atmosphere. To each vial was added 50 μl of 0.05 M buspirone solution in 1:1 trifluoroacetic acid/acetonitrile sparged with nitrogen stream. Reaction mixture irradiated with Kessil 34 W blue LED for 18 hr using EvoluChem photochemical device.

PhotoRedOx Flow Reactor

PhotoFlowReactorRedOx_Box_interchim_blog0218The common limitation to scaling up photoredox chemistry is due to the low penetration of the light in to the reaction mixture (few mm) which prohibits the use of large reaction vessels. Surface area is key to shorten reaction time. It is possible to significantly increase the surface area by running the reaction in flow. This will decreases the reaction time and allows to be run in continuous mode for scale-up.

To solve this challenge, we designed a flow reactor that can be used in the PhotoRedOx Box. This flow reactor is using PFA tubing and has volume of 2 ml. Comparing reactions in flow and in batch we observed significant decrease in reaction time.

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Reaction protocol

In a 4-ml vial equipped with a Teflon septa were weighed NiCl2-dme (1.1 mg, 5 μmol, 0.05 mol %) and dtbbpy (1.3 mg, 5 μmol, 0.05 mol %). 1 ml of dry MeOH was added to the vial and the vial was stirred on an orbital shaker until complete dissolution. The solution was evaporated to dry at room temperature. Then Ir(dF-CF3-ppy)2(dtbpy) (1.1 mg, 1 μmol, 0.05 mol %), and 4-bromoacetophenone (9.95 mg, 100 μmol, 1 equiv.) were added. 1 ml of dry acetonitrile was added followed by Et3N (21 μmol, 300 μmol, 3 equiv.) and aniline (4.65 mg, 100 μmol, 1equiv.). The solution was sparged with nitrogen via submerged needle for 5 minutes.
Several batches of 100 μl of solution were successively injected to the flow reactor placed in EvoluChem PhotoRedOx Box with blue Kessil LED using an injection module (Gilson) and the samples were circulated using a HLPC pump at different flow rates to allow residence time of 5, 10, 15, 20 and 30 min. Reaction completion was monitored by LC-MS using the ratio bromoacetophenone/product.

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SchemaPhotoRedOx-FR-5_interchim_blog0218

Reaction protocol

In a 4-ml vial equipped with a Teflon septa were weighed NiCl2-dme (1.1 mg, 5 μmol, 0.1 mol %) and dtbbpy (1.3 mg, 5 μmol, 0.1 mol %). 1 ml of dry MeOH was added to the vial and the vial was stirred on an orbital shaker until complete dissolution. The solution was evaporated to dry at room temperature. Then Ir(dF-CF3-ppy)2(dtbpy) (1.1 mg, 1 μmol, 0.1 mol %), and 4-bromoacetophenone (4.98 mg, 50 μmol, 1 equiv.) were added. 1 ml of dry acetonitrile was added followed by 2,6 lutidine (17.5 μmol, 150 μmol, 3 equiv.) and potassium benzyltrifluoroborate (9.90 mg, 50 μmol, 1 equiv.). The solution was sparged with nitrogen via submerged needle for 5 minutes.
Several batches of 100 μl of solution were successively injected to the flow reactor placed in EvoluChem PhotoRedOx Box with blue Kessil LED using an injection module (Gilson) and the samples were circulated using a HLPC pump to allow residence time of 30 min. Reaction completion was monitored by LC-MS using the ratio bromoacetophenone/product.

To know more :

Acces directly to our products dedicated to PhotoRedox on our website.



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