LEGO MINDSTORMS Fraction Collector: A low-cost tool for a Preparative High Performance Liquid Chromatography System

Abstract

Preparative High Performance Liquid Chromatographic (prep-HPLC) systems are used in many research schemes including purifying products from reaction mixtures, fractionating natural product extracts, and isolating compounds. Manual fraction collection from a prep-HPLC is a common method; however, it often lacks the reproducibility of automated fraction collectors due to human error. Automated fraction collectors for prep-HPLC systems range can add thousands of dollars to the cost of prep-HPLC and are thus not always available to budgetary constrained research programs. Nevertheless, an automated fraction collector is a tremendous resource for any lab that employs prep-HPLC methods. Using LEGO MINDSTORMS pieces and easily obtained lumber and steel C-channel we were able to deploy an automated fraction collector for only a fraction of the cost of a commercial instrument. The programming software allows for a simple interface to create fraction collection programs tailored to individual HPLC methods. This fraction collector can be connected to any LC system and tailored to collect fractions in nearly any size or shaped container. This fraction collector was designed to provide maximum versatility and will make automated fraction collection more accessible to all researchers. The simple interface allows for quickly adapting the fraction collector method to any liquid chromatographic separation, no matter how complex.

Graphical Abstract

Introduction

Preparative High Performance Liquid Chromatography (prep-HPLC) is an extremely powerful separation technique that has developed rapidly over the past 50 years¹. It is used for the separation or isolation of compounds from complex mixtures. This now common technique is routinely used to isolate and refine high-purity compounds from reaction mixtures, natural product extracts, and amino acids^{2, 3}. It is also the main technique for the separation of chiral compounds⁴. Additionally, fractionation of complex mixtures for further refinement or biological testing can be performed by prep-HPLC⁵. HPLC methods utilize the wide variety of high quality chromatography columns available; these columns with robust HPLC method development can yield highly specific separations that are extremely repeatable.

In the case of complex fractionation or complex separations, very low yields of refined compounds often result. One solution to low yield, is repetitive prep-HPLC runs. Manual collection of the HPLC effluent is often the only method available; however, the process is highly labor-intensive and involves the risk of human error. An automated fraction collector that can be adapted to many LC systems and accommodates varied fraction sizes is of great utility. However, commercial fraction collectors for preparative HPLC systems can cost thousands of USD even for refurbished equipment and significantly more for new units. This cost is in addition to the prep-HPLC system itself and the assorted chromatography columns. High initial costs and maintenance of equipment can become a significant burden for smaller research groups, scientists working under tight budgetary constraints, in resource-limited environments, and in primarily undergraduate institutions. Thus, a fraction collector made with less costly materials, such as LEGO pieces and hardware found at home improvement stores, can greatly ease the economic burden for researchers.

The LEGO MINDSTORMS® system was built as an “intelligent brick” 15 years ago that could be programmed by a computer⁶. It can be used to create a variety of robots and experience microchip programming. In educational settings, it has been implemented to teach engineering concepts^{7, 8} as well as construct laboratory equipment such as liquid handling systems⁹ and a colorimeter¹⁰. Broad availability of the MINDSTORMS system and LEGO pieces has enabled the dissemination of ideas and designs using easily accessible platforms, such as YouTube.

The MINDSTORMS system is controlled by the EV3 Programming software which is free and multiplatform; available for Windows, Mac, Android and iOS. The EV3 software can connect to MINDSTORMS projects over a USB cable or Bluetooth wireless connections. In most instances, once the MINDSTORMS project is uploaded into the controller, the connection is no longer required. All software can be downloaded from the LEGO website¹¹. Additionally, secondary software and websites have been built to purchase accessories and create LEGO designs. The basic MINDSTORMS set includes the intelligent EV3 brick (controller), three servo motors, a variety of LEGO pieces and sensors; the MSRP is $349.99 USD. The fraction collector detailed here makes use of the basic LEGO MINDSTORMS set, LEGO Technic pieces, standard LEGO bricks, metal pipes, lumber and steel C-channel materials. The total price of all materials was less than $500 USD. These materials allowed the construction of an automated prep-HPLC fraction collector, which can be used with nearly any sized fraction containers, at a much lower cost than a commercial fraction collector. This design plan can easily be adapted to meet a wide variety of research needs and connected to any LC system or even gravity columns.

Materials and Methods

Building the Robot

This fraction collector robot was built around the MINDSTORMS EV3 system. However, the servos and controller could be adapted to many microchip platforms, such as Arduino¹². The main components of the fraction collector consist of: (1) a wooden Base holds the fraction bottles with a steel C-channel and round tubing frame attached to the base providing rails that (2) the Motor Assembly and fraction collector arm travel along, and (3) the HPLC Delivery Tube Assembly which moves along the fraction collector arm. The Supporting Information contains general construction details, brick-by-brick instructions drawn using BrickLink Studio 2.0¹³, and example fractionation programs. A YouTube video outlining the fraction collector assembly and demonstrating its use is available at: https://youtu.be/LzYv31-Kuns.

The construction of the fraction collector can be further broken down into sub-assemblies to assist in construction. The wooden Base and Risers should be constructed first. Then the steel C-channel Frame of the Motor Assembly which also serves as the fraction collector arm (Fig. 1a). The Frame is then surrounded by and affixed into a LEGO Case (Fig. 1b–d). The Motor Assembly Unit (Fig. 1e, f) is attached to the bottom of the Case and Frame and contains the motors and pulleys which move the assembly back and forth along the rails. The Control Unit (Fig. 1g) contains the MINDSTORMS Intelligent EV3 Brick (microcontroller) and sensors. After assembly of the major sub-assemblies, a medium motor is mounted to the front of the Case to control the HPLC Delivery Tube Assembly.

Figure 1.

Photos of the fraction collector sub-assemblies: a) Metal Frame, b) top view of the LEGO Casing Unit for the Metal Frame, c) bottom view of the Casing Unit, d) top cover of the Casing Unit, e) top view of the Motor Assembly Unit, f) side view of the Motor Assembly Unit, g) EV3 Control Unit with sensors, h) medium motor.

Programming the LEGO MINDSTORMS System

The MINDSTORMS EV3 Programming software is downloaded from the LEGO website¹¹. The LEGO website has tutorials on how to program the EV3 system and there are many additional videos on YouTube. This fraction collector can only collect fractions by time. However, in the authors’s experience this is not a limitation. If the method was already run on the prep-HPLC system, elution times for the fractions can be determined from this past run. If the method was developed on an analytical HPLC system using a column with the same chemistry, scaling the run to the prep-HPLC system is straightforward¹⁴. The size and number of fraction bottles or tubes will change the spacing on the fraction collector bed, thus altering the rotation of the motor of the robot’s fraction collector arm, so the fraction collector program must be adapted for different sizes and spacing of bottles.

The basic method for the fraction collector is to collect by time using three program loops; (loop 1) move the HPLC Delivery Tube away from the Control Unit, collecting fractions in a row, (loop 2) then move the fraction collector arm over one row, and (loop 3) moving the HPLC Delivery Tube back toward the Control Unit, collecting fractions in this next row. Each loop runs for the allotted amount of time and turns the motor on the fraction collector arm a specified number of degrees which would vary based on the dimensions of the collection bottles. Each loop is then repeated to move the fraction collector through the total number of bottles. This basic program loop can be adapted to collection for different times and many bottle types. Additionally, there is a prestart sequence, which allows for the flushing of the Prep-HPLC system and holding the fraction collection until the HPLC run begins. A post-run sequence is added after the fractions are collected to move the fraction collector arm into a waste bottle.

More complex fraction collection schemes are also possible. For example, a series of 60 second fractions, followed by one 5 min. fraction, and then ending with a series of 3 min. fractions. Two example fractionation methods are described in the Supporting Information.

Connection to a Prep-HPLC System

This fraction collector can connect to any HPLC system, flash chromatographic system, or even open gravity columns. The outlet tubing from the LC’s detector is connected directly into the fraction collector using a long piece of PTFE LC tubing, which is coiled around the fraction collector arm and inserted into the HPLC Delivery Tube Assembly. Specific assembly instructions are in the Supporting Information.

In order to begin the fraction collector program concurrently with the HPLC method, a MINDSTORMS touch sensor was placed on the front of the prep-HPLC. A pause in the program holds the HPLC Delivery Tube in the waste position until “start” button is pressed. Then the fraction collector arm moves to begin collecting fractions according to the program. A video demonstration of the assembly and a prep-HPLC run is available at https://youtu.be/LzYv31-Kuns. Alternately, the analogue start signal of the HPLC system can be connected to one of the EV3 controller analogue inputs to automatically trigger the beginning of a run. After using the fraction collector with the “start button” it was determined that this wiring was not necessary for the authors’s needs.

Discussion and Conclusions

Following programming and connection to an Agilent Technologies 1260 Infinity II preparative HPLC System, repetitive HPLC runs were performed with the LEGO MINDSTORMS fraction collector robot depositing multiple HPLC runs into the designated square 500 mL French bottles in effort to isolate substantial material for further analysis (Fig. 2).

Figure 2.

HPLC chromatogram of iterative HPLC separations using the customized fraction collector. Collections can be programmed to be variable in number and duration. The example shown here displays fractions ranging from 60 seconds to 360 second fractions. Maximum fraction size is limited only by collection vessels and flow rate.

Preparative HPLC is a commonly used separation technique in many chemical isolation schemes. Manual fraction collection is possible with any chromatographic separation; however, the addition of an automated fraction collector adds a high degree of repeatability to the chromatography and frees the researcher to focus on other tasks. The use of widely available materials, including the LEGO MINSTORMS kit, at relatively affordable prices, allows the addition of a powerful, highly customizable be style fraction collector to any research program. This design can be adapted to work with many bottle sizes and scaled to nearly any size to accommodate the needs of any laboratory. While the EV3 software for the LEGO MINDSTORMS system requires basic programming skills, these can be learned very quickly via free online resources and by modifying this articles example programs. After learning how to write programs using the EV3 software the fraction collector can be adapted for nearly any chromatographic separation. The ease of construction and affordability of this fraction collector robot should make this powerful instrument available to any research or educational program.