{'fraction_non_alphanumeric': 0.030923327636233556, 'fraction_numerical': 0.013176199371994117, 'mean_word_length': 4.544182459233142, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
The Assembly and Use of Continuous-Flow Systems for Chemical Synthesis

The adoption and opportunities in continuous-flow chemical synthesis (“flow chemistry”) have increased significantly over the past several years. Nevertheless, while several continuous-flow platforms are commercially available, ‘in-lab’ constructed systems provide researchers with a flexible, versatile, and cost-effective alternative. Herein, we describe how to assemble modular continuous-flow apparatus from readily available and affordable parts in as little as 30 minutes. Furthermore, our experiences in single- and multi-step continuous-flow synthesis have been documented to provide solutions to commonly encountered technical problems. Following this Protocol, a non-specialist can assemble a continuous-flow system from reactor coils, syringes, in-line liquid-liquid separators, drying columns, back pressure regulators, static mixers, and packed bed reactors.


INTRODUCTION
This century has observed an overwhelming advancement in the understanding and synthesis of organic compounds.
In contrast, laboratory technology has remained much the same; reactions are still labor intensive and performed in round bottom flasks housing stir bars and reflux condensers. Although this style of chemistry (termed batch chemistry) is often suitable for chemical transformations, increasing research has demonstrated that continuous flow can provide a swathe of benefits. [1][2][3][4][5][6][7][8][9] Translating reactions into continuous flow can provide a level of control and automation not possible in batch reactions. Improving the heat and mass transfer, 10,11 micromixing, 12 radiation penetration, [13][14][15][16] and homogeneity 6 of reactions has provided practical solutions to improve reaction efficiencies. Moreover, continuous flow allows the safe handling of hazardous species. Generating toxic compounds in situ for immediate consumption in sequential reactions avoids stockpiling risk-prone compounds. 1,4,8 Harnessing increased reaction efficiencies, environmental sustainability, and improved safety metrics has lead to the adoption of continuous flow into both academia and industry. 3,[17][18][19][20][21] At its core, continuous-flow synthesis is achieved by pumping solutions of reactants through reactors in a continuous fashion (Fig. 1A). For lab-scale purposes, syringe, HPLC, or peristaltic pumps drive fluid through reactor coils, or microfluidic chips, with the residence time of the fluid dictated by the total flow rate and reactor volume.
Although single reactor coils are commonly utilized, advancements of in-line technology have provided opportunities to explore more complex, multi-step transformations. 6,22 In this context, in-line refers to a manipulation, or analysis, of the continuous-flow stream without the solution exiting the continuous-flow system. The ability to perform aqueous workups, drying, and other purification procedures has also advanced continuous-flow notably.
Multi-step continuous-flow systems have provided rapid syntheses of active pharmaceutical ingredients (APIs) and natural product intermediates as well as effective functional group transformations. 3,9 What started as a niche discipline in academia around ten years ago has evolved into a rapidly diversifying and expanding research area. Furthermore, materials, 23-26 biochemical [27][28][29][30] and biofuels [31][32][33][34] processes have been translated into continuous-flow systems with success. In 2005, Lonza Ltd. stated that ~50% of chemical processes could benefit from translation into continuousflow. 35 In this respect, it is vital that all researchers wishing to contribute, and advance this area, have access to readily available and operationally simple continuous-flow equipment.
Although continuous-flow chemistry has a plethora of benefits, it has drawbacks and limitations as well. For example, handling solids in continuous-flow is difficult due to reactor clogging. Though advancements in reaction engineering and immobilized reagents have overcome this to a certain extent, further innovation is needed. The most challenging constraints for academics, however, remain the high costs and significant time investment needed to conduct continuous-flow research. 13 First, pumping modules are by far the most expensive component of a continuousflow system ($1000-20,000 USD). Second, purchasing commercially available reactors and equipment increases the cost of continuous-flow synthesis immeasurably. Furthermore, researchers are often reliant on long delivery periods for equipment. Although syringe pump costs are fixed, the other constraints can be overcome through the assembly of modular continuous-flow systems from commercially available and affordable components (Fig 1A). This not only reduces the time and cost associated with continuous-flow chemistry, but opens up continuous-flow chemistry to all. subsequent moisture sensitive transformations. Combining these modules into specific orders allows researchers to translate a large number of synthetic protocols into continuous-flow systems.
Our laboratory strives to ensure that continuous-flow apparatus is (i) modular, so it can be easily removed and replaced to afford a variety of systems, (ii) simple and quick to assemble from readily available and cost effective parts, and (iii) easy to operate to allow both new, and experienced researchers to harness the benefits of continuous-flow chemistry. For example, the synthesis of lidocaine hydrochloride requires assembling multiple syringes, pumps, reactor coils, a back pressure regulator, and an in-line liquid-liquid separator (Fig. 1B). To then synthesize ibuprofen, users need only to rearrange the syringe pumps, add a new reactor coil, and then exchange a static mixer for a back pressure regulator. This whole process takes ~30 minutes and allows the synthesis of another API.
Overview of the procedure Figure 2| Outline of the equipment constructed in this protocol. First the assembly of a basic continuous-flow set up is detailed from procedure i-iii. Then, the assembly of advanced continuous-flow flow equipment is detailed from procedure iv-vii.
In this protocol, the assembly, use, and troubleshooting of modular continuous-flow systems are discussed. For reader convenience, the protocol has been split into two sections. The first describes the assembly of basic continuousflow equipment. The second describes the incorporation of advanced in-line technologies for more complex syntheses.
For basic continuous-flow systems, the assembly of reactor coils from PFA tubing and the assembly of stainless steel syringes are discussed. Finally, the assembly of a continuous-flow system housing a back pressure regulator, two syringes, and a single reactor coil is demonstrated. For those new to continuous flow, this is the most basic set up. The skills learned in this section, however, can be applied to the assembly of more complex systems in future syntheses.
In the advanced section, the assembly, inclusion, and use of in-line technology will be described to allow more complex, multi-step continuous-flow transformations (Fig. 1B). First, the assembly of a static mixer and a packed bed reactor for increased mixing of solutions is discussed. Second, the use of a liquid-liquid separator for intermediate purification is detailed. Finally, how to assemble a drying unit housing molecular sieves is demonstrated. This protocol allows an undergraduate researcher to quickly assemble reactor coils, static mixers, stainless steel syringes, packed bed reactors, back pressure regulators, in-line liquid-liquid separators and drying units. Compiling this equipment into a simple modular continuous-flow system allows those new to the field to explore continuous-flow synthesis. 1| Cut DuPont ® 0.02" inner diameter PFA tubing to the required length using polymer tube cutters. Use the equations found in the supplementary information to calculate the reactor volume required for a specific residence time at a desired flow rate. CRITICAL STEP Use polymer tube cutters to cut the PFA tubing to reduce the chance of reactor leaking. Polymer tube cutters provide a flush cut ensuring optimal sealing of the fitting.
2| Insert one end of the PFA tubing through a super flangeless nut™, ensuring that the thread of the nut is pointing towards the open end of the PFA tubing.
3| Slide the stainless steel ring of the ferrule set for 1/16" outer diameter tubing onto the PFA tubing using tweezers.
Ensure the slanted part of the stainless steel ring is facing towards the open end of the PFA tubing, and the thicker portion towards the nut ( Fig. 3A and D). CRITICAL STEP If the stainless steel ring is installed backwards, the reactor will not seal properly and, as a consequence, can leak.
4| Slide the yellow plastic component of the ferrule set for 1/16" outer diameter tubing onto the PFA tubing using tweezers. Ensure the cone portion of the yellow plastic component is facing the super flangeless nut™ and the stainless steel ring ( Fig. 3B and D). CRITICAL STEP If the yellow plastic component is installed backwards, the reactor will not seal properly and, as a consequence, can leak.
5| Insert the end of the PFA tubing into a low-pressure PEEK union for 1/16" outer diameter tubing. Ensure the PFA tubing is constantly pushed into the low-pressure union while finger tightening the super flangeless nut™ (Fig.   3C). CRITICAL STEP If the PFA tubing is not constantly pressed into the union during tightening, the tubing may not be aligned with the ferrule during compression. This can lead to reactor failure and leaking.
6| Further tighten the super flangeless nut™ into the low-pressure union using the extender tool for standard head nuts. CRITICAL STEP Do not over tighten the ferrule as this will lead to deformation and/or cracking of the nut.
This can lead to reactor failure and leaking.
7| Unscrew the flangeless nut from the union using the extender tool for standard head nuts. Ensure that the ferrule set is flush with the PFA tubing. CRITICAL STEP If this is not perfect, cut off the ferrule and the super flangeless nut™ and redo. Be mindful that cutting the tubing will decrease the residence time of the fluid in the reactor. ? TROUBLESHOOTING 8| Repeat steps 2-7 on the other end of the PFA tubing.
9| Coil the PFA tubing into a circle with a diameter less than the bath used to mediate the temperature of the reactor coil (Fig. 3E).
10| Use two cable ties to fasten opposite sides of the reactor coil into shape (Fig. 3F).
11| Finish the reactor coil by cutting the cable ties to a workable length (Fig. 3G). Stainless steel syringes are ideal for those new to the field of continuous-flow chemistry. Some reagents however are not compatible with stainless steel, and in this case, glass or plastic syringes should be used. It is worth noting that plastic and glass syringes are more affordable, but using stainless steel syringes allows fluid delivery at higher pressures (>25 bar). Furthermore, glass syringes can shatter, and plastic syringes can crack if the system clogs, posing a safety concern. Although stainless steel syringes require greater financial investment, their long-term use and wide operational ability is highly advantageous. Accordingly, this part of the protocol describes the assembly of stainless syringes for use in a continuous-flow system. Here, 8 mL stainless steel syringes used due to their broad solvent capacity compared to the 20 mL stainless steel syringe; this relates to O-ring degradation. Furthermore, 8 ml syringes are capable of dealing with higher-pressure systems, and as a consequence, fail less frequently. If a 20 mL stainless steel syringe is required, ensure that perfluoroelastomer barrel O-rings are installed (Harvard Apparatus, #PY2 5013-089).
1| Unpack the contents of the stainless steel syringe kit (Fig. 4A) Cut a 30 cm long piece of DuPont ® 0.02" inner diameter PFA tubing and ensure both ends have been cut flat with a polymer tube cutter. CRITICAL STEP Use polymer tube cutters to cut the PFA tubing to reduce the chance of reactor leaking. Polymer tube cutters provide a flush cut ensuring optimal sealing of the fitting.
4| Pass the PFA tubing through the metal nut (component 1) ensuring the threaded side of the nut is facing the end of the PFA tubing (Fig. 4D).
5| Slide the stainless steel ring (component 2a) onto the tubing using tweezers. Ensure the thicker side of the stainless steel ring is facing towards the nut, and the thinner side facing the end of the PFA tubing. Next, slide the stainless steel cone (component 2b) onto the PFA tubing using tweezers. Ensure the larger diameter side of the stainless steel cone is facing the metal nut and the stainless steel ring, and the smaller diameter face is pointing towards the end of the PFA tubing (Fig. 4D). Note that the Swagelok ferrules do not sit flush with the tubing as described for the super flangeless nut™ and ferrule in the previous section. Swagelok ferrules sit a few mm onto the PFA tubing.

CRITICAL STEP
If not correctly constructed the syringe can leak.
6| Insert the open end of the PFA tubing into the syringe connector (component 3). Ensure the PFA tubing is constantly pushed forced into the syringe connector and tighten the nut by hand (Fig. 4E). CRITICAL STEP If the PFA tubing is not constantly pushed into the syringe connector while tightening, the depth of the Swagelok ferrules may be incorrect and reactor leaking can occur.
7| Using two 5/16" wrenches to further tighten the metal nut onto the syringe connecter. CRITICAL STEP Do not over tighten as this can lead to constriction of the PFA tubing, and in extreme cases can break the PFA tubing. A properly tightened ferrule will remain in position when the nut has been disconnected from the syringe connector, but does not pinch tube PFA tubing.

(iii) Assembly of a basic continuous-flow system TIMING 30 min
This part of the protocol describes the assembly and operation of a simple continuous-flow system harnessing two syringe pumps at an elevated pressure. First, the pressurization of a Zaiput variable back pressure regulator is discussed. A variable back pressure regulator offers reaction flexibility, and in our experience, lasts longer than other back pressure regulators, but other options are available such as the fixed back pressure regulator cartridges from IDEX Health and Science (#P-795 and P-764 for example). Operating under a back pressure allows solvents to be heated above their atmospheric boiling points while providing a smooth flow of fluid through the system. Second, the assembly of a continuous-flow system housing two syringe pumps, a T-mixer with check values, a single reactor coil and a back pressure regulator is detailed. This set up allows two solutions to be mixed, reacted, and then collected for analysis. Although a simple system, the skills in this section are invaluable for the assembly of more complex systems.
(a) Pressurization of the Zaiput variable back pressure regulator TIMING 10 min (supplemental video

3)
1| Unpack the contents of the variable back pressure regulator kit to reveal a back pressure regulator and a gas delivery tube.
2| Attach the gas delivery tube to a tank of compressed gas housing an adjustable regulator with a needle valve (Fig.   5A). Note that air is typically used, but N 2 or argon can also be used.
3| Set the compressed gas regulator pressure to the desired pressure to be programmed into the variable back pressure regulator. For example, 100 psi of compressed air equals a back pressure of 100 psi in the variable back pressure regulator (Fig. 5B).
4| Fully open the valve on the back pressure regulator with a 1/10" screwdriver in an anti-clockwise motion. This will vent all the gas from the back pressure regulator setting the pressure to atmospheric (Fig. 5C). CRITICAL STEP Failure to vent the previous pressure within the back pressure regulator may lead to a higher than intended pressure due to prior pressure carrying over.
5| Connect the end of the gas delivery tube to the back pressure regulator and tighten by hand (Fig. 5C).
6| Fully open the needle valve on the compressed gas tank and allow pressure equilibration into the back pressure regulator for ten seconds. CRITICAL STEP Failure to allow full equilibration will lead to an inaccurate back pressure being used.
7| Using a 1/10" screwdriver, close the valve on the back pressure regulator by screwing in a clockwise motion (

(b) Assembly of a generic continuous-flow system TIMING 20 min (supplemental video 4)
1| Cut a 15 cm long piece of DuPont ® 0.02" inner diameter PFA tubing using polymer tube cutters to ensure both ends have been cut flat. CRITICAL STEP Use polymer tube cutters to cut the PFA tubing to reduce the chance of reactor leaking. Polymer tube cutters provide a flush cut ensuring optimal sealing of the fitting.

2|
To one end of this tubing add a super flangeless nut™ and a ferrule set as previously described in steps 2-7 of reactor coil assembly. ? TROUBLESHOOTING 3| Insert the super flangeless nut™ from the PFA tubing into the outlet port of the pressurized back pressure regulator. First tighten the nut using the extender tool for standard head nuts, and then using pliers. CRITICAL STEP Make sure the tubing is attached to the correct port of the back pressure regulator as indicated by the arrow (Fig. 6A). Furthermore, over tightening the super flangeless nut™ can lead to PFA tubing damage and cracking of the super flangeless nut™.
4| Gather the previously made reactor coil. Screw a PEEK T-junction mixer with 0.02" inner diameter to one end of the reactor coil. Tighten the nut using the extender tool for standard head nuts (Fig. 6B). CRITICAL STEP Over tightening the super flangeless nut™ can lead to PFA tubing damage and cracking of the super flangeless nut™.

5|
Attach the other end of the reactor coil to the back pressure regulator (Fig. 6D). To do this, insert the super flangeless nut™ from the reactor coil into the remaining inlet port of the back pressure regulator. First tighten using the extender tool for standard head nuts, and then use pliers. CRITICAL STEP Over tightening the nut can lead to PFA tubing damage and cracking of the nut. nuts™ with the extender tool for standard head nuts, and then pliers (Fig. 6E). Load the stainless steel syringes into the syringe pumps ready for infusion (Fig. 6F). It is recommended to tighten all nuts used in the continuousflow system with pliers before system initiation. ? TROUBLESHOOTING 8| If the reactor is to be heated or cooled, place the reactor coil into a bath, and reach the required temperature. In 12| Wash the stainless steel syringes with water and soap and then dry with acetone. Before using again ensure that Krytox grease has been added to the O-rings of the stainless steel syringe plunger.
13| Disconnect the reactor coil from the back pressure regulator and remove from the bath. Lay the reactor on paper towels to absorb the oil and unscrew the PEEK T-junction mixer.
114| Blow compressed air through the reactor coil to remove residual solvent.

Advanced Procedures Improved mixing in a continuous-flow system
After the use of a basic continuous-flow system, more intense mixing may be required. Although solutions mix efficiently in the PEEK T-junction mixer, some situations generate large slugs of two immiscible fluids (slug flow).
Further micromixing through a static mixer or packed bed reactor can increase reaction efficiencies. In the first instance, incorporate a static mixer after the PEEK T-junction mixer, and before the reactor coil. However, if still ineffective, pass the fluid through a packed bed reactor housing sand, stainless steel, or glass beads. Packed bed reactors provide efficient and sustained mixing, and have been utilized in several continuous-flow syntheses. 36 This section of the protocol describes assembly of a static mixer and packed bed reactor housing sand.
CRITICAL STEP Use polymer tube cutters to cut the PFA tubing to reduce the chance of reactor leaking.
Polymer tube cutters provide a flush cut ensuring optimal sealing of the fitting.
Remove the Teflon static mixer from its packaging (Fig. 7A) and insert into the PFA tubing using tweezers (Fig.   7C). ! CAUTION The static mixer is sensitive and too much pressure will bend and snap it -be careful.
3| Using the end of the tweezers, gently push the Teflon static mixer down the PFA tubing until a headroom of ~1 cm is visible (Fig. 7D). If only a small static mixer is required, then proceed to the next step. If more extensive mixing is required, add more static mixers on top of each other to create a larger mixing region. If more than three static mixers are required we suggested try a packed bed reactor (see below). ? TROUBLESHOOTING 4| Add a ferrule set for 1/8" outer diameter tubing (component 2 and 3, Fig. 7B) and a super flangeless nut™ for ≤1/8" outer diameter tubing (component 1, Fig. 7B) to the PFA tubing using tweezers. Ensure that the orientation of the components is the same as shown in Fig. 7E. Tighten and secure the ferrule set as previously described in steps 2-7 of reactor coil assembly. 2| Mount the seamless stainless steel tubing into a bench vise and mark out a 10 cm portion (Fig. 8B). Place the metal tube cutter around the point to be cut, and tighten. Rotate the metal tube cutter around the stainless steel tubing and at every ten turns, tighten the tube cutter further to ensure the blade of the tube cutter remains in contact with the stainless steel tubing (Supplemental video 6). Keep rotating the metal tube cutter around the stainless steel tubing until cut. If the metal tubing is moving then the tube cutters are to tight. In this case, loosen the tube cutters and tighten less every ten turns.
3| File both ends of the cut stainless steel tubing flat using a metal file (Supplemental video 7). Then, deburr the opening using a deburring tool to ensure the inside surface is flush with the rest of the stainless steel tube.
Push the frit to the bottom using tweezers, and then reattach the metal nut (Fig. 8E). CRITICAL STEP Inserting the frit into the 1/4" ports (component 1) at this stage avoids incorrect spacing in step 5.

5|
To both ends of the stainless steel tubing add the Swagelok union parts in the order and orientation shown in Fig. 8C. 6| Compress the components and tighten by hand in a clockwise motion to secure. Mount the stainless steel tubing into the bench vise and use 1/2" and 9/16" wrenches to tighten the nut into the Swagelok union further. CRITICAL STEP This needs to be tight; the packed bed mixer is under pressure.
7| Unscrew the smaller metal nut (component 0) and lay the contained ferrule set out as shown in Fig. 8D. Be careful to not lose the components inside, they can sometimes slide out.
8| Cut 2 x 10 cm long pieces of DuPont ® 0.02" inner diameter PFA tubing with a polymer tube cutter to ensure both ends of the tubing are flat. These will serve as the tubing connecting the packed bed mixer to the other parts of the continuous-flow system. CRITICAL STEP Use polymer tube cutters to cut the PFA tubing to reduce the chance of reactor leaking. Polymer tube cutters provide a flush cut ensuring optimal sealing of the fitting. 12| To both ends of the PFA tubing add a super flangeless nut™ and ferrule set using the method previously described in steps 2-7 in the reactor coil assembly section (Fig. 8I).
13| Unscrew the metal nut (component 2) from the Swagelok union (component 1) to reveal an opening into the stainless steel tube.

14|
To create a funnel for loading, take a 3 mL disposable plastic syringe and completely remove the plunger. Insert the small end of the syringe into the stainless steel tube so that it is held firm.
15| Add sand into the stainless steel tube while simultaneously tapping the stainless steel tubing and the syringe with a spatula to ensure good packing and sand release (Supplemental video 9). Other materials can be used in the packed bed reactor such stainless steel chippings, however, use of very small packing materials can cause a large pressure increase leading to syringe pump failure. A general rule is to use a material that will require ten units or more to span the diameter of the stainless steel tubing. For example, if the internal diameter of the packed bed reactor is 0.180", then sand particles with a diameter of ≤0.0180" should be used.
16| Use the flat back end of a drill piece to pat down the sand to ensure complete and firm packing.
17| Place you finger on top of the filled stainless steel tubing and subject the surrounding nuts to compressed air to remove any loose sand. CRITICAL STEP Sand caught in the metal nut can lead to an inefficient seal.
18| Screw the metal nut (component 2) into the Swagelok union (component 1) using 1/2 and 9/16" wrenches to ensure a tight fitting. The packed bed reactor is now complete and ready to use. To change the material in the packed bed reactor, repeat steps 13-18 ensuring the sand is disposed into solid waste and that the column is washed with a solvent of choice. Unpacking the column is best when the material contained within the column is dry. ?

(b) In-line purification of intermediate compounds
In multi-step continuous-flow transformations, the purification of intermediate compounds is vital to ensure high reaction yields. Liquid-liquid extractions can be achieved in-line by injecting water into the continuous-flow stream through a T-mixer and then directing the stream into a liquid-liquid separator. Here, the organic phase passes through a semi-permeable membrane and enters new PFA tubing. The aqueous phase does not pass through the membrane, and enters separate PFA tubing. If water-soluble salts or impurities are present they will remain in the aqueous phase, purifying the organic stream to some extent. The organic phase (or aqueous phase) can be channeled into another reactor coil for a sequential reaction, or can be collected. This next section describes the assembly and cleaning of a Zaiput liquid-liquid separator.

(vi) Use of an in-line liquid-liquid separator TIMING 30 min
If using a new in-line liquid-liquid separator proceed to step 11.
1| Remove the six screws (three per side) from both sides of the separator using a 3/32" Allen key as depicted in 4| Unscrew the metal disk surrounding the diaphragm with a 5/64" Allen key (Fig. 9B).
5| Carefully remove the diaphragm with tweezers. CRITICAL STEP Damaging this part will cause the separator to fail.
6| Wash all of the components and the device with soapy water, ethanol, and finally acetone. Leave the device to dry for a few hours, using a paper towel to remove the majority of the water.
7| Place a new precut porosity membrane from Zaiput Flow Technologies onto the device as depicted in Fig. 9E.
Alternatively, sheets of porosity membrane can be purchased, and then cut in to the correct size using a scalpel.

CRITICAL STEP
The rough side of the membrane must face upwards. If this membrane is placed on the component with the seal the wrong way up, separation can fail.
8| Insert the diaphragm (a new one if the old one is damaged) back into the metal disk, ensuring that when it is placed back into the device the raised face is pointing into the concave part of the separator (Fig. 9D).
9| Screw the metal disk into place, being careful not to over tighten the screws by using the cross tightening pattern.
10| Place the lid of the separator into place and insert all six screws with a 3/32" Allen key. Tighten each screw gently to ensure an even fit by using the cross tightening pattern.
11| Cut 3 x 10 cm pieces of DuPont ® 0.02" inner diameter PFA tubing and cut both ends using polymer tube cutters.
CRITICAL STEP Use polymer tube cutters to cut the PFA tubing to reduce the chance of reactor leaking.
Polymer tube cutters provide a flush cut ensuring optimal sealing of the fitting.
12| Attach super flangeless nuts™ and ferrule sets to both ends of the PFA tubes using the method previously described in steps 2-7 of the reactor coil assembly section.
13| Attach the PFA tubes to the three separator inlets. Insert the super flangeless nut™ into the entry of the separator and tighten with pliers to ensure a tight fitting (Fig. 9F). CRITICAL STEP These super flangeless nuts™ have to be tight otherwise the separator will leak.
14| Attach a 20 psi back pressure regulator to the aqueous stream and an 21 psi charged back pressure regulator to the organic stream as depicted in Fig. 9G. CRITICAL STEP These values are not absolute and depending upon the continuous-flow setup, values will vary. In the first instance, if just using a separator at the end of the system, see if separation is achieved with no back pressure on either of the exiting streams. Importantly, the pressure on both sides must be near equal. Thus, if the organic side leads into a packed bed reactor and a drying unit, this will add pressure to the organic side and must be counteracted by placing a back pressure on the aqueous side. ? TROUBLESHOOTING 15| Flow through toluene (50 mL) and then water (50 mL) to equilibrate the device at a flow rate of 500 µL/min. CRITICAL STEP Pre-charging the membrane in this manner will improve the separation efficiency.  2| Unpack the contents of an Omnifit chromatography column kit (150 mm length). Remove the retaining cap from the column, and fully extend the adjustable end piece (Fig. 10A). 3| Cut 2 x 15 cm pieces of DuPont ® 0.02" inner diameter PFA tubing and cut flat the ends of the tubing with a polymer tube cutter. CRITICAL STEP Use polymer tube cutters to cut the PFA tubing to reduce the chance of reactor leaking. Polymer tube cutters provide a flush cut ensuring optimal sealing of the fitting.

4|
To both pieces of 15 cm PFA tubing attach a blue plastic nut with a ferrule set (contained in the column kit, but a super flangeless nut™ can also be used) to one end of the tubing, and a super flangeless nut™ with a ferrule set to the other end of the PFA tubing using the method previously described in steps 2-7 in the reactor coil assembly section (Fig. 10D).
5| Attach one of the 15 cm PFA tubes to the connection cap (found in the column kit) by inserting the blue plastic nut from the PFA tubing into the blue connection cap and tightening using pliers. Attach the connection cap to the fully extendable end piece and tighten (Fig. 10F).
6| Thread the nut from the extendable end piece onto the glass column. Turn in a clockwise motion until firm, but not tight (Fig. 10F).
7| Twist the upper portion of the nut counter clockwise to lower the variable length section. Once the desired depth has been achieved, fully tighten the lower nut onto the glass column in a clockwise fashion. To remove the extendable end piece, reverse steps 6 and 7.
8| Attach the other 15 cm PFA tube to a connection cap by inserting the blue plastic nut from the PFA tubing into the blue connection cap and tightening using the pliers. Attach the connection cap to the retaining cap and tighten ( Figs. 10C and B).
9| Fill the chromatography column to 95% volume (130 mm deep) with 4 Å molecular sieves directly from the oven and immediately cap with the retaining head Figs. 10H. ! CAUTION Use laboratory grade oven gloves as this glass beaker is extremely hot. We suggest clamping the column in an upright position to make this easier (Fig.   10G).
10| Screw the retaining head into the column until firm. ! CAUTION Over tightening will cause the glass column to crack.

EXAMPLE REACTION
The reaction below is used in our laboratory to teach continuous-flow synthesis. In this experiment, 4chlorobenzenesulfonyl chloride 1 and dibenzylamine 2 react to yield sulfonamide 3 in a single reactor with an in line quench (Fig. 11). Several skills including: reactor construction, charging of a back pressure regulator, the assembly of stainless steel syringes, the assembly of a continuous-flow system with multiple junctions, and yield determination are described. standard head nuts and then pliers (Fig. 12). CRITICAL STEP Over tightening the nut can lead to PFA tubing damage and cracking of the nut.
4| Place the reactor coil into an oil bath and secure the exit and entry of the reactor coil by use of binder clips (Fig.   12F). Ensure that the silicone oil covers the reactor to ensure even heating. CRITICAL STEP Make sure that the binder clips are not pressing upon the reactor tubing, this can lead to a clog.
5| Secure the first PEEK Y-assembly mixer by holding it in place with a clamp (Fig. 12F).
6| Secure the back pressure regulator with a clamp (Figs. 12 C and F). CRITICAL STEP Ensure the exit of the reactor is attached to inlet of the back pressure regulator in the orientation as shown in Fig. 12F.
7| Using Part 1 "Assembling of a reactor coil", assemble a 10 cm long reactor from 0.02" inner diameter DuPont ® tubing with super flangeless nuts™, and ferrule sets for 1/16" outer diameter tubing on both ends.
8| Insert one end of the 10 cm reactor into the exit of the back pressure regulator and finger tighten. Further tighten this joint with the extender tool for standard head nuts and then pliers (Fig. 12E). CRITICAL STEP Over tightening the nut can lead to PFA tubing damage and cracking of the nut.

9|
Insert the other end of the 10 cm reactor into a second PEEK Y-assembly mixer for 1/16" outer diameter tubing, 0.60" thru hole and finger tighten (Fig. 12E). Further tighten this joint with the extender tool for standard head nuts.
CRITICAL STEP Over tightening the nut can lead to PFA tubing damage and cracking of the nut.
10| Using Part 1 "Assembling of a reactor coil", assemble a 20 cm long reactor from 0.02" inner diameter DuPont ® tubing with super flangeless nuts™, and ferrule sets for 1/16" outer diameter tubing.
11| Insert one end of the 20 cm reactor into the second PEEK Y-assembly mixer and finger tighten (Fig. 12E). Further tighten this joint with the extender tool for standard head nuts. CRITICAL STEP Over tightening the nut can lead to PFA tubing damage and cracking of the nut.
12| Insert the other end of the 20 cm reactor into a 25 mL glass beaker; this will serve as the collection vial during system equilibration. 3| Add anhydrous DCM to both flasks using a disposable 10 mL plastic syringe and a hypodermic needle under typical air free conditions to create ~9 mL final volume solutions (Fig. 12A).
4| Add 1/2" PTFE stir bars to both solutions A and B and stir at 300 rpm for 10 min at RT. This will completely dissolve 2 and ensure mixture homogeneity (Fig. 12B). Adjust the volumes of both solutions to 10 mL by adding anhydrous DCM using a disposable 10 mL plastic syringe and a hypodermic needle under typical air free conditions. stainless steel syringe to the first PEEK Y-assembly mixer at the entry of the reactor and finger tighten (Fig. 12C).

CRITICAL STEP
Further tighten this joint with the extender tool for standard head nuts. CRITICAL STEP Over tightening the nut can lead to PFA tubing damage and cracking of the nut. and attach the tubing from the exit of the stainless steel syringe into the second PEEK Y-assembly mixer at the end of the continuous-flow system (Fig. 12E). Further tighten this joint with the extender tool for standard head nuts.
CRITICAL STEP Over tightening the nut can lead to PFA tubing damage and cracking of the nut.
8| Before starting the experiment, ensure that all connections are secure and that the stainless steel syringes are properly secured into the Harvard syringe pumps.

(iii) Performing the experiment TIMING 40 min
1| Set the temperature of the oil bath to 80 °C and the stir rate to 400 rpm. Wait until the temperature of the oil bath is stable for at least ten min.
2| Set both Harvard syringe pumps to infuse at 125 µL min -1 , and start the infusion. 10| Add ~700 mg of MgSO 4 to the organic layer and swirl for 2 min to dry the solution. The solution will achieve a snow globe effect once dry. If 700 mg of MgSO 4 does not achieve this effect, add small portions until achieved.
11| Filter the solution into a 250 mL round bottom flask using a glass powered funnel and qualitative filter paper.
12| Carefully remove the organic solvent under reduced pressure using a rotatory evaporator; a white power will result.
13| Take a small quantity of this solid (~1 mg) and add it to 5 mL of ethyl acetate. Perform TLC analysis of the product (R f =0.368) using 1:10 ethyl acetate: hexanes against both 4-chlorobenzenesulfonoyl chloride 2 (R f =0.500) and dibenzylamine 1 (R f =0.205). The TLC should indicate a single product under UV radiation (Fig. 12D).
15| Combine the fractions containing the desired product and remove the solvent under reduced pressure using a rotary evaporator. Dissolve the resulting solid into a small amount of DCM (~5 mL), and transfer to a pre-weighed 25 g vial.

16|
Remove the solvent under reduced pressure using a rotatory evaporator until dry. Further dry the product under vacuum at 40 °C for 2 h. Weight the 25 g vial to obtain the mass of the product. The theoretical yield for this transformation is 177.5 mg.  The components were not correctly oriented on the PFA before tightening.

Isolated
Attach a new ferrule set in the order, and orientation, as shown in Figure 3A and B. Do not reuse ferrule set. The ferrule was not around the PFA tubing when the super flangeless nut™ was tightened into the union, or the super flangeless nut™ was not tightened enough.
When tightening the super flangeless nut™ apply pressure to the PFA tubing as shown in Figure 3C to ensure the ferrule is set around the PFA tubing. Do not reuse ferrule.
Procedure ii, Step 7 The PFA tubing disconnected from the syringe connector.
The ferrule set was not around the PFA tubing when tightened.
When tightening the metal nut into the syringe connector, apply pressure to the PFA tubing to ensure the ferrule is set around the PFA tubing. Procedure ii, Step 8 The PFA tubing disconnected from the super flangeless nut™ after tightening.
The components were not correctly oriented on the PFA before tightening.
Attach a new ferrule set in the order, and orientation, as shown in Figure 3A and B. Do not reuse ferrule set. The ferrule was not around When tightening the super flangeless nut™ the PFA tubing when the super flangeless nut™ was tightened into the union, or the super flangeless nut™ was not tightened enough. apply pressure to the PFA tubing as shown in Figure 3C to ensure the ferrule is set around the PFA tubing. Do not reuse ferrule.
A failure of the Swagelok to PFA connection is common. Mechanical stress leads to the stainless steel biting into the PFA tubing.
This region of the tubing should be inspected before every use. It should be replaced if wear is observed.
The syringe plunger has become sticky and hard to operate when being filled with fluid.
The grease has been removed from the O-rings.
Ensure regulator maintenance is conducted. Grease the O-rings of the syringe plunger with Krytox grease during maintenance.
Procedure iiia, Step 8 The gas delivery tube detaches from the compressed air tank valve at high pressures.
The gas delivery tube is not secure.
Tighten the metal nut at the end of the gas delivery tube to ensure a firm fit between the back pressure regulator and the gas delivery tubing. Procedure iiia, Step 9 The back pressure regulator does not allow fluid though when not connected to a flow system (i.e. stand alone) The device is broken.
Contact Zaiput Flow Technologies for advice. The fluid from the continuous-flow system is entering the wrong side of the separator.
Switch the entry and exit tubes of the back pressure regulator. The engraved arrow on the back pressure regulator shows the direction of fluid flow. Procedure iiib, Step 2 The PFA tubing disconnected from the super flangeless nut™ after tightening.
The components were not correctly oriented on the PFA before tightening.
Attach a new ferrule set in the order, and orientation, as shown in Figure 3A and B. Do not reuse ferrule set. The ferrule was not around the PFA tubing when the super flangeless nut™ was tightened into the union, or the super flangeless nut™ was not tightened enough.
When tightening the super flangeless nut™ apply pressure to the PFA tubing as shown in Figure 3C to ensure the ferrule is set around the PFA tubing. Do not reuse ferrule.
Procedure iiib, Step 7 The use of air or moisture sensitive reagents is required; How do I load them into the stainless steel syringes under appropriate conditions? Stainless steel syringes require the reagents to be drawn up by the user, so this has to be carried out under air-free conditions.
(a) Prepare the solutions to be flowed through the continuous-flow system as you would for typical syntheses, i.e. in a round bottom flask or screw top vial. Next, screw a quick-stop luer in-line check valve (IDEX Health & Science, #P-655) to the super flangeless nut™ of the stainless steel syringe and join a suitable needle. Using this needle, the solutions can be loaded into the syringes using the same method as you would use to remove anhydrous solvent from a solvent bottle (standard air sensitive syringe techniques). (b) Syriss Asia pumps remove the need to transfer solutions from schlenk flasks into syringes. Attaching a quick-stop luer in-line check valve and needle to the pump allows direct infusion into the continuous-flow system. However, these pumps cost ~$20,000 USD. (c) To note is that if reagents are highly air sensitive, then PFA tubing allows slow permeation of air and moister. This will lead to poor yields. Use stainless steel tubing for the reactor coil. Procedure iiib, Step 8 Reactor coil does not submerge in the oil bath.
Orientation or buoyancy of the reactor is wrong.
Clip the entrance and exit of the reactor coil to the oil bath. Ensure the orientation of the reactor coil leaves it fully submerged in oil/ fluid. Make sure the clips do not press upon the reactor tubing as this can cause a clog. Furthermore, a lead weight can be used to weight down the coil in the bath. A reactor temperature of over 150 °C is required or the PEEK mixers must be submerged in the oil bath.
PEEK nuts and PFA tubing can fail at higher temperatures.
Use stainless steel mixers and tubing to handle extreme temperatures (IDEX Health & Science, #U-428). The manufacturer recommends a PFA tubing temperature limit of 80 °C. We have used PFA tubing at 200 °C under 170 psi without fail, although, safety should be the prime concern here. To note, if PEEK fittings are heated and cooled subsequently, the connections must be re-tightened or leaking will occur. If using high reactor temperatures, we suggest using smaller inner diameter tubing (0.01" or 0.02") given the thicker tubing wall. Syringe pumps stopped injecting the fluid into the continuous-flow system.
The continuous-flow system is clogged and/or the syringes can only delivery fluids up to a certain pressures.
This limit depends upon the type of pump used.
(a) Working backwards from the last piece of apparatus in the continuous-flow system, unscrew the apparatus and see if the syringe pumps are able to inject. This will help identify where the clog is. (b) If solid formation is located in the reactor coil replace the reactor coil or declog the reactor. Then, try diluting the reaction, placing the reactor coil into a sonicating bath, or using larger diameter tubing to handle solids. If increasing the internal diameter of the tubing we suggest using mixers with the same internal diameter thru holes. (c) If the packed bed reactor is clogged, change the stainless steel frits to ones with larger porosities (IDEX Health & Science). Furthermore, we have experienced that using stainless steel and other small diameter packing materials can induce an unworkable back pressure. Switching to sand maintains great mixing, but lowers the pressure the packed bed reactor infers on the system. (f) If solid formation occurs in the back pressure regulator, sonicate the solution or dilute the reaction before it enters the back pressure regulator. If the back pressure regulator is clogged, disassemble to clear. Solids in back pressure regulator can perforate the diaphragm. Contact Zaiput Flow Technologies for replacement diaphragm and parts. (g) If solid formation is occurring in the union connecting two parts of the continuous-flow system, use a larger internal diameter union or dilute the reaction. (h) If solid formation is occurring in the inline liquid-liquid separator, try another solvent, and/or dilute the reaction. (i) Solutions can react in the syringe causing precipitation. Check for solid formation in the syringe. If present, separate the reactive components into separate syringes. (j) The length of the continuous-flow system is to long for the capabilities of the syringe pumps. Shorten the length of the continuous-flow system, or remove the apparatus inferring large pressures (i.e. packed bed reactors housing stainless steel chippings). (k) Check the pressure ratings of the syringe pumps. Harvard syringes have a factory force level of 50%; change this to 100%. Syrris syringe pumps have a factory setting of 15 bar; change to 25 bar. (l) The mixers have become blocked due to solid formation upon mixing. Change the mixers to have larger inner diameter channels. For example, change to a 0.06" thru T-mixer. If the problem persists, trial a range of mixers, then proceed to a tube-intube mixer. (m) If the continuous-flow stream is viscous, dilute with solvent to decrease back pressure. Syringe pump has broken.
Although far less likely that a reactor clog, sometimes syringe pumps break and need to be repaired.
Send the pump to be repaired.
The PFA tubing in the continuous-flow system burst.
The pressure of the system is too high for the specific PFA tubing. The system could have also been heated, or cooled, beyond the limit of the specific tubing. Lastly, the tubing could be damaged due to mechanical stress such as kinking.
(a) If using larger diameter tubing than 0.02" inner diameter, change to this smaller diameter tubing; the smaller diameter tubing has a thicker wall making it more resistant to higher pressures and temperatures. To note is that 0.01" inner diameter tubing is available for a more extreme option. (b) Switch to stainless steel tubing for the reactor coil. This is constructed much the same way a packed bed reactor is. (c) Ensure the reactor is not kinked or twisted. Solid formation can lead to a clog. This will in turn increase the pressure of the system leading to a rupture of the PFA tubing.
Reaction engineer the transformation so that solid formation is avoided or limited. This could be through dilution, variation in the reagents, or changes to the reaction temperature.
The reaction releases a large volume of gas that dramatically increases the system pressure.
(a) Dilute the reaction. (b) Lower the back pressure regulator to decrease the total system pressure. (c) If gas release is due to reagent degradation, contemplate different reaction conditions such as a lower reaction temperature.
Lower yields are observed when the continuous-flow system gets longer or more complex.
In general, a larger system has higher pressures, thus, if operating at low flow rates (~10 µl/min) the syringe driver can slip, lowering the amount of reagent added to the continuous-flow system. This will reduce the yield.
Use higher flow rates to avoid the syringe slipping at lower flow rates. An increase in the length of the reactor coils will have to occur for the residence time to stay the same. Furthermore, ensure the syringe size is correctly inputted into the syringe pump.
Long pieces of PFA tubing between back pressure regulators, packed bed reactors, T-mixers and drying units can create complex pressure gradients. Instead of flowing in one direction it can recycle around. This can lead to lower yields or over reactivity.
(a) Reduce the dead volume in the system. Have the shortest amount of PFA tubing possible between apparatus. (b) If adding several solvents at different Tmixers, combine all these solvents together into one stream for mixing with the primary continuous-flow stream. This means that instead of several T-mixers, there is only one, reducing the possibility for complex pressure gradients.
Procedure iv, Step 3 Using three static mixers does not provide enough mixing.
Static mixers only provide a certain level of mixing.
Switch to a packed bed reactor housing sand, stainless steel chippings or glass beads. This will increase the amount of micromixing in the system. As mentioned previously, be careful of the back pressure this infers on the system. Procedure v, Step 10 The part labeled 0 in Figure 8A snaps when the nut is tightened. This is the weakest point of the packed bed reactor. Over tightening will cause it to bend and snap.
Be careful when tightening this joint. Ensure the wrench is at 90° to the packed bed reactor when tightening. Proceed carefully.
Procedure v, Step 18 When tightening the nut between part 2 and part 1 in Figure 8A, a crunching noise is heard.
There is sand present in joint.
While covering the entrance to the packed bed reactor, tap the reactor to loosen sand or other materials. Subject the nut to compressed air to blow away loose debris.

Procedure vi, Step 11
The separator is not separating the organic and aqueous layer.
There is too much pressure on the stream where no fluid is exiting.
Change the back pressure regulators to either lower the pressure on the side where no fluid is exiting, or increase the pressure on the side where the fluid is exiting. Through this process there will be ideal pressures for optimal separation. The membrane is fouled or is compromised with solids.
Replace the membrane.
The pore size of the membrane is to small or to large for the flow rate being used.
Change to a different diameter pore size membrane. The specific type is specified in the instructions.
The flow rate going through the separator is too high.
Lower the flow rate or use a higher capacity separator. The organic layer is miscible with water.
Add a non-miscible organic solvent for the extraction process.