Name: tuning a parallel segmented flow column and enabling multiplexed detection
Uploaded: 2015-12-15T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Tuning a Parallel Segmented Flow Column and Enabling Multiplexed Detection at JoVE.com

This work was supported by UWS and ThermoFisher Scientific. One of the authors (DK) acknowledges the receipt of an Australian Postgraduate Award.

Note: This protocol contains instructions on how to use a PSF column on a HPLC system coupled with multiple detectors for multiplexed detection. The protocol has been written assuming the reader has basic knowledge and experience in chromatography and various HPLC detection methods.
Caution: Please refer to material safety data sheets (MSDS) for all materials and reagents before use (i.e., MSDS for methanol). Ensure the use of all appropriate safety practices when handling solvents and High Performance Liquid Chromatography (HPLC) eluent. Ensure appropriate use of engineering controls of HPLC, analytical balance and detector instrumentation, and ensure the use of personal protective equipment (safety glasses, gloves, lab coat, full length pants, and closed-toe shoes).
1. Setup of HPLC Instrument
<ol>
	<li>Prepare the HPLC instrument with ultrapure water (e.g., 100% Milli-Q water) for line A and 100% methanol for line B as the mobile phase and purge the pumps as per manufacturer requirement. If one of the detectors used is MS, as it is here, add 0.1% formic acid to both mobile phases A and B.</li>
	<li>Set up the HPLC instrumental components and detectors as illustrated in Figure 2. This requires convenient placement of detectors relative to the column so as to minimize the dead volume between detector and column. Flexibility in the HPLC system configuration is desirable.</li>
</ol>
2. Setup of UV-Vis and MS Detectors
<ol>
	<li>Set the UV-Vis detector to the desired wavelength dependent on the sample of interest (e.g., 280 nm).</li>
	<li>Set the MS detector in positive mode for Total Ion Count (TIC) analysis using Full Scan detection method. Also adjust the following MS parameters accordingly: vaporizer temperature 500 &#176;C, capillary temperature 350 &#176;C, sheath gas set at a rate of 60 units, auxiliary gas flow 40 and sweep gas flow at 5 units, and spray voltage 3.5 kV. These settings can be adjusted later for specific user requirements dependent on the sample being analyzed.</li>
</ol>
3. Preparation of 2,2-Diphenyl-1-picrylhydrazyl Radical (DPPH&#8226;) Reagent Setup of DPPH&#8226; System Detector
<ol>
	<li>Weigh 25 mg of DPPH&#8226; and dissolve in 250 ml of methanol in a volumetric flask.</li>
	<li>Add 250 &#181;l of formic acid to the DPPH&#8226; reagent. Cover the flask in foil to prevent exposure to light.</li>
	<li>Sonicate the flask containing the DPPH&#8226; reagent for 10 min.</li>
	<li>Purge the DPPH&#8226; pump with the prepared DPPH&#8226; reagent as per manufacturer&#39;s requirement.</li>
	<li>Set up the DPPH&#8226; system according to Figure 2 by attaching the pump line to the inlet of a T-piece.</li>
	<li>Attach a 100 &#181;l reaction coil to the outlet of the T-piece and attach the other end of the reaction coil to the detector.</li>
	<li>Encase the reaction coil in a column heater and set the column heater temperature to 60 &#176;C.</li>
	<li>Set the DPPH&#8226; UV-Vis detector to 520 nm.</li>
</ol>
4. Setup of PSF Column
<ol>
	<li>Connect the inlet of the PSF column to the HPLC instrument.</li>
	<li>Connect the central port to the MS detector, using a 15 cm length of 0.13 mm i.d. tubing.</li>
	<li>Connect a peripheral port to the UV-Vis detector using a 15 cm length of 0.13 mm i.d. tubing.</li>
	<li>Connect another peripheral port to the T-piece of the DPPH&#8226; detection system using a 15 cm length of 0.13 mm i.d. tubing.</li>
	<li>Block the unused peripheral port using a column stopper.</li>
	<li>Bring the flow rate of the HPLC pump to 1 ml min-1 at 100% line B - 100% Methanol (0.1% formic acid).</li>
	<li>Equilibrate the column with the 100% methanol mobile phase for 20 min for a 4.6 mm i.d. x 250 mm length column. This time is scaled according to the dimensions of other columns the user may employ.</li>
</ol>
5. Tuning the PSF Column for Multiplexed Detection
<ol>
	<li>Measure the mass of at least two empty collection vessels (one for the port connected to the UV-Vis detector and one for the DPPH&#8226; detector) using an analytical balance.</li>
	<li>Collect mobile phase from the UV-Vis and the DPPH&#8226; ports into two separate, pre-weighed collection vessels (5.1). Record the period of time for the collection. Collect at least 500 mg of solvent in each vessel.</li>
	<li>Weigh the collection vessels and determine the mass of mobile phase. Given the density of methanol is 0.791 g ml-1, determine the volume of mobile phase collected from each port.</li>
	<li>By difference, that is, nominal pump set flow rate minus the flow rate through the DPPH&#8226; and UV-Vis detector flow ports, determine the flow rate to the MS detector. Express each flow proportion as a percentage of the total flow. 
	Note: Ideally, the flow percentages are: to the MS is 18% of the total flow rate, to the UV-Vis 22%, to the DPPH&#8226; detector 60%.</li>
	<li>If not, adjust the flow percentages by changing the back pressure on the UV-Vis detector. For example, if the flow to the UV-Vis is too high, decrease the proportion by adding add a 15 cm section of 0.13 mm i.d. tubing to the outlet of the UV-Vis detector. Then repeat steps 5.1 to 5.5.</li>
</ol>
6. Final Setup Conditions
<ol>
	<li>Set the flow rate of the DPPH&#8226; reagent pump to the same flow rate exiting the outlet port connected to the DPPH&#8226; detector. 
	Note: The PSF column multiplexed with UV-Vis, DPPH&#8226; and MS is now ready for analysis.</li>
</ol>

<ol>
	<li>Camenzuli, M., Ritchie, H. J., Ladine, J. R., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+design+of+a+new+concept+chromatography+column.">The design of a new concept chromatography column.</a> Analyst. 136 (24), 5127-5130 (2011).</li><li>Camenzuli, M., Ritchie, H. J., Ladine, J. R., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+separation+performance+using+a+new+column+technology:+Parallel+segmented+outlet+flow.">Enhanced separation performance using a new column technology: Parallel segmented outlet flow.</a> J. Chromatogr, A. 1232, 47-51 (2012).</li><li>Camenzuli, M., Ritchie, H. J., Ladine, J. R., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+flow+management+in+preparative+chromatographic+separations:+A+preliminary+investigation+into+enhanced+separation+using+a+curtain+flow+inlet+fitting+and+segmented+flow+outlet.">Active flow management in preparative chromatographic separations: A preliminary investigation into enhanced separation using a curtain flow inlet fitting and segmented flow outlet.</a> 35 (3), 410-415 (2012).</li><li>Camenzuli, M., Ritchie, H. J., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradient+elution+chromatography+with+segmented+parallel+flow+column+technology:+A+study+on+4.6mm+analytical+scale+columns.">Gradient elution chromatography with segmented parallel flow column technology: A study on 4.6mm analytical scale columns.</a> J. Chromatogr., A. 1270, 204-211 (2012).</li><li>Camenzuli, M., Ritchie, H. J., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+HPLC+separation+performance+using+parallel+segmented+flow+chromatography.">Improving HPLC separation performance using parallel segmented flow chromatography.</a> Microchem. J. 111, 3-7 (2013).</li><li>Shalliker, R. A., Ritchie, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segmented+flow+and+curtain+flow+chromatography:+Overcoming+the+wall+effect+and+heterogeneous+bed+structures.">Segmented flow and curtain flow chromatography: Overcoming the wall effect and heterogeneous bed structures.</a> J. Chromatogr, A. 1335, 122-135 (2014).</li><li>Camenzuli, M., Ritchie, H. J., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+active+flow+technology+HPLC+columns+as+a+platform+for+multiplexed+detection.">Evaluating active flow technology HPLC columns as a platform for multiplexed detection.</a> Microchem. J. 110, 473-479 (2013).</li><li>Camenzuli, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+segmented+outlet+flow+high+performance+liquid+chromatography+with+multiplexed+detection.">Parallel segmented outlet flow high performance liquid chromatography with multiplexed detection.</a> Anal. Chim. Acta. 803, 154-159 (2013).</li><li>Shalliker, R. A., Camenzuli, M., Pereira, L., Ritchie, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+segmented+flow+chromatography+columns:+Conventional+analytical+scale+column+formats+presenting+as+a+'virtual'+narrow+bore+column.">Parallel segmented flow chromatography columns: Conventional analytical scale column formats presenting as a 'virtual' narrow bore column.</a> J. Chromatogr., A. 1262, 64-69 (2012).</li><li>Soliven, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+performance+of+narrow-bore+HPLC+columns+using+active+flow+technology.">Improving the performance of narrow-bore HPLC columns using active flow technology.</a> Microchem. J. 116, 230-234 (2014).</li><li>Camenzuli, M., Ritchie, H. J., Dennis, G. R., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+segmented+flow+chromatography+columns+with+multiplexed+detection:+An+illustration+using+antioxidant+screening+of+natural+products.">Parallel segmented flow chromatography columns with multiplexed detection: An illustration using antioxidant screening of natural products.</a> Microchem. J. 110, 726-730 (2013).</li></ol>

The authors have nothing to disclose.

This study involves the characterization and profiling of coffee using HPLC with multiplexed detection employing a parallel segmented flow (PSF) column. Multiplexed HPLC using PSF columns enables the characterization and identification of key chemical entities by reducing the data complexity of the sample whilst obtaining a greater degree of molecule-specific information within a fraction of the time it takes using conventional multi-detection processes. The PSF column not only allows a platform for multiplexed detection...

Active Flow Technology Columns
Active flow technology (AFT) chromatography columns were recently developed to overcome inefficiency in separations associated with flow heterogeneity1-6 and also to enable multiplexed detection. In this particular communication we detail the operational process of the parallel segmented flow (PSF) column with multiplexed detection. The key functional advantages of the PSF column are: (1) the flow from the radial central region of the column bed is isolated from the peripheral or wall region of flow, (2) the volume of mobile phase that must be processed by a detection source is reduced, and (3) detection sources can be multiplexed to expand sample information without instilling detection delays across each detection process, or subsequently necessitating the splitting of a post column flow stream7,8. The key feature in the design of the PSF column that enables the advantage of multiplexed detection is the novel outlet fitting and frit assembly. Figure 1 is a photograph of the AFT column compared to a conventional column. It is important to understand that the splitting process obtained using parallel segmented flow columns is not the same as post column flow stream splitting. In a post column flow stream split the entire sample band from the leading edge to the extremities of the tail is sampled equally (i.e., axially), thereby, each flow stream is equal with respect to efficiency and sensitivity; the magnitude of the sensitivity thereby being divided by the number splits. In PSF, however, the splitting process samples the band radially, not axially. As such, the central port samples the peak apex &#8212; the most concentrated region of the peak. Thus, the sensitivity here is highest as the peak is not diluted by the diffuse tailing region. The sample eluting from the peripheral ports is not as efficient as in the central zone, but, since the band is sampled radially, rather than axially, the width of the peak is narrower than would be the case for a sampling process that divides the peak in the axial direction, i.e., a post column split. Hence the sensitivity with a concentration dependent detector is not reduced.
In the PSF column, the outlet fitting comprises multiple exit ports and on the inside of this end fitting there is housed an annular frit. The inner portion of this annular frit channels flow out of the column via the radial central outlet port, while the outer radial portion of the outlet frit channels flow out of the column via the peripheral or wall region outlet flow ports. The inner and outer portions of the outlet frit are separated by an impermeable barrier that prevents cross flow between these flow regions2. As a consequence of this design the central radial flow stream through the column bed is separated from the wall region flow inside the column. The relative portion of flow from these two regions can be varied to almost any desired ratio through pressure management in order to optimize various functional aspects of the column technology, such as separation efficiency or detection sensitivity. In essence, this design effectively establishes within the larger format column a &#39;virtual&#39; column, having a narrower internal diameter, and hence the column functions as a true wall-less column, overcoming column bed heterogeneity and the wall effects9,10.
The major benefits of PSF columns are improvements in column efficiency, minimization of the solvent processing for the detection source(s) and enabling multiplexed detection. However, an added advantage is that since the tailing and fronting regions of any band are removed from the overall elution profile the solute at elution or detection is present in a higher concentration than would otherwise be observed for the same solute injection and concentration load on a conventional column, depending on the segmentation ratio employed. As a consequence there is often observed a gain in signal intensity for separations conducted on PSF columns2. In fact, if the segmentation ratio is adjusted such that 25% of the flow exits from each of the four exit ports, the signal intensity that is observed using Ultra Violet (UV) detection shows practically the exact same signal intensity as apparent using a conventional column where the entire (100%) of the mobile phase is analyzed7. Furthermore, fine tuning of the outlet ratio between the central and wall flow regions allows the column efficiency to be optimized. The gains in column efficiency observed using AFT columns cannot be stated to a single value, since these efficiency gains are a function of three factors: (1) the flow rate, (2) the segmentation ratio, and (3) the solute retention factor. Nevertheless, gains in efficiency compared to conventional columns are almost always observed, and sometimes these gains are more than 100% in the number of theoretical plates1,2. The ability to tune the segmentation ratio allows the analyst to effectively tailor the diameter of the &#39;virtual&#39; column, and this is an important factor in relation to the detection process. For example, a virtual 2.1 mm internal diameter (i.d.) column is established from a physical 4.6 mm i.d. column when the segmentation ratio is 21% of mobile phase eluting from the radial central exit port. Under these conditions, the virtual 2.1 mm i.d. column performs with an efficiency that may be more than 70% greater than the conventional 2.1 mm i.d. column, depending on flow rate, and solute retention factor10.
The current PSF column design that is used for multiplexed detection incorporates a 4-port outlet fitting, but the column can be fitted with a 2-port end-fitting also, however, this limits detection to only two detectors. The basic operation of these columns is, however, the same, except that four detectors can be coupled simultaneously to the 4-port outlet PSF column broadening the scope for multiplexed detection. Aside from pre- and post- column connective tubing, the only additional requirements to operate a PSF column is tubing that can be connected to the peripheral outlet ports, and a means by which the amount of mobile phase passing through each tube can be measured, typically either a mass measurement or a volumetric measurement. For ease of tuning, the internal diameter of all outlet flow tubing should be the same. The flow ratio between the peripheral and radial central exit ports is then varied through the use of pressure management, simply by changing the length of the tubing located on the peripheral outlet fitting, or the length of tubing post detector on the radial central outlet port.
Multiplexed Detection Using PSF Columns 
An important advantage of PSF columns is that each of the outlet exit ports can be connected directly to a detection source, thus enabling multiplexed detection. In a well-designed detection system a single analysis with multiplexed detection can provide substantial information in regards to the nature of the components within the sample. Importantly, destructive and non-destructive tests can be conducted at exactly the same time, without detection delay. This allows the absolute assignment of, for example, anti-oxidants using DPPH&#8226; reagent, with components observed to elute with UV and/or mass spectrometry (MS) detection responses7,11. Therefore, four independent detectors can be operated simultaneously with appropriate portions of flow directed to each detector via any of the four outlet ports. Since the flow through these ports can be easily adjusted the amount of solute reaching any of the detectors can be adjusted to suit the sensitivity of the given detector source. It should be noted, however, that the most efficient solute migration is observed through the radial central outlet port. Each of the peripheral ports offer equivalent separation efficiency, which when set to 25% through each port, is only slightly less efficient than a conventional column. As such, it is important that the quantitative detector be set to analyze sample from the radial central exit port.
When setting up a PSF column for the purpose of multiplexing detection there are a number of considerations that need to be made to achieve efficient and high quality results; that is tube dimensions for each port, choice of which port for a type of detector and flow adjustment.
Tube Dimensions for Each Port
In chromatography the length of post column tubing plays a crucial role in the efficiency and performance of the separation. Large dead-volume as a result of long or wide i.d. tubing from column outlet to detector will result in a loss of efficiency, resolution and sensitivity. Thus, appropriate tubing dimensions must be utilized when setting up the PSF column to attain the maximum potential in providing efficient separation whilst providing the benefits of multiplexing.
Port to Detector 
Figure 2 is an illustration of an example setup of multiplexed detection (Ultra Violet-Visible (U-Vis), mass spectrometer (MS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH&#8226;) detection). The illustration shows the central port is attached to the MS detector, whereas the DPPH&#8226; and UV-Vis detectors are attached to the peripheral ports. Since the MS is the most sensitive detector of the three, flow to this detector was directed from the central outlet port. As DPPH&#8226; detection is selective to the presence of antioxidants, and the least sensitive and most tolerant to band broadening, flow to this detector was directed from a peripheral port. The UV-Vis was a secondary &#39;generic&#39; detector, so flow to this detector was directed from a second peripheral port.
Flow Adjustment
Once the appropriate tubing has been attached from a port to detector, the exiting flow from each of the detectors can be adjusted to the required amount. A simple way to measure the amount of flow exiting from each detector is to weigh the amount of mobile phase that elutes through each port over a given period of time. The percentage flow can thus be determined, and the flow ratios can be adjusted by either shortening or lengthening the tubing attached to the outlet line on the detectors accordingly to suit the requirements of the detectors of choice. Different detectors have different requirements of flow, for example, the flow cell of a fluorescence detector (FLD) is not flow rate limited, but care must be taken to avoid over-pressurization of the flow cell. Hence the control of flow through the FLD is usually attained by adjusting the pressure drop across the other detectors and the remainder of the flow then passes through the FLD. A detector that is sensitive to the amount of flow that is delivered is the MS. Generally, current high end mass spectrometers can readily process around 1 to 1.5 ml/min of moderately aqueous mobile phase. Above this flow rate, flooding of the source may make the MS inoperable. However, detection sensitivity in most mass spectrometers is benefited by using lower flow rates; hence the PSF flow splitting capabilities are extremely useful for applications involving MS detection. High column volumetric flow rates can be utilized, but with low volume loads transported to the MS detector. Tuning of the flow to the MS detector, however, must be made by adjusting the pressure drop prior to the MS detector, rather than post MS. Here, the use of narrow bore tubing (0.1 mm i.d.) is very useful, as the pressure can be readily adjusted without adding inappropriate dead volume.
Depending on the type of detector the adjustment of the segmentation ratio can be done either pre or post detector. If a non-destructive detector, such as UV-Vis is used, the flow percentage would be measured and tuned post detector. If a single destructive detector is used in the multiplex set up the flow percentage is determined by back calculating with respect to other port flow percentages. If a reagent based detector is used such as DPPH&#8226;, the flow percentage is measured post detector without the addition of reagent; and if two or more destructive detectors are used, then the flow ratio is measured pre-detector. Detection systems that may require additional instrumentation, such DPPH&#8226;, will have extra system pressure that may alter the flow percentage once attached to the detection system. Therefore, careful consideration should be paid to system pressure of a destructive detector, when adjusting flow percentage pre-detector. Irrespective of the flow ratio that is set through any of the ports, quantitative information should be obtained through appropriate standardization. Once the flow ratios are set, however, they are robust, and they do not change even under gradient elution conditions7,
The detailed video protocol accompanying this manuscript is intended to show how to use and tune a PSF column functioning in a multiplexed mode of detection.

<table><tbody><tr><td>HPLC instrument</td><td></td><td></td><td>Multiple detectors of choice for multiplexed detection. Detectors of choice may require additional instrumentation, &lt;em&gt;e.g.&lt;/em&gt;, pump.</td></tr><tr><td>Parallel Segmented Flow HPLC column</td><td>Thermo Fisher Scientific</td><td>Not Defined</td><td>Soon to be commercialized</td></tr><tr><td>Methanol</td><td>Any brand</td><td></td><td>HPLC Grade</td></tr><tr><td>PEEK tubing</td><td>Any brand</td><td></td><td>Various lengths and i.d.</td></tr><tr><td>Column stoppers</td><td>Any brand</td><td></td><td>For blocking unused peripheral ports.</td></tr><tr><td>PEEK tube cutter</td><td>Any brand</td><td></td><td></td></tr><tr><td>Analytical Scale Balance</td><td>Any brand</td><td></td><td></td></tr><tr><td>Stop watch</td><td>Any brand</td><td></td><td></td></tr><tr><td>Eluent collection vessels</td><td>Any brand</td><td></td><td>1-2 ml sample vials can be used as eluent collection vessels</td></tr></tbody></table>

tuning a parallel segmented flow column and enabling multiplexed detection

Active flow technology (AFT) is new form of column technology that was designed to overcome flow heterogeneity to increase separation performance in terms of efficiency and sensitivity and to enable multiplexed detection. This form of AFT uses a parallel segmented flow (PSF) column. A PSF column outlet end-fitting consists of 2 or 4 ports, which can be multiplexed to connect up to 4 detectors. The PSF column not only allows a platform for multiplexed detection but also the combination of both destructive and non-destructive detectors, without additional dead volume tubing, simultaneously. The amount of flow through each port can also be adjusted through pressure management to suit the requirements of a specific detector(s). To achieve multiplexed detection using a PSF column there are a number of parameters which can be controlled to ensure optimal separation performance and quality of results; that is tube dimensions for each port, choice of port for each type of detector and flow adjustment. This protocol is intended to show how to use and tune a PSF column functioning in a multiplexed mode of detection.

A multiplexed HPLC analysis was carried out using an AFT column in PSF mode (Figure 1) and set up as illustrated in Figure 2. This type of setup allowed a coffee sample to be analyzed simultaneously using UV-Vis, DPPH&#8226; and MS in Total Ion Count (TIC) mode. The compounds of the coffee sample that responded to DPPH&#8226; could then be easily matched up to the UV-Vis and MS - TIC responses based on the alignment of retention time as illustrated in Figure...

Watch this Scientific Journal Video about Tuning a Parallel Segmented Flow Column and Enabling Multiplexed Detection at JoVE.com

Tuning a Parallel Segmented Flow Column and Enabling Multiplexed Detection

Here, we present a protocol for the operation and tuning of parallel segmented flow chromatography columns to enable multiplexed detection.

Active flow technology (AFT) is new form of column technology that was designed to overcome flow heterogeneity to increase separation performance in terms ...

tuning-parallel-segmented-flow-column-enabling-multiplexed

Research

JoVE Journal

Chemistry

7.3K Views.  University of Western Sydney. Here, we present a protocol for the operation and tuning of parallel segmented flow chromatography columns to enable multiplexed detection. 

Video: Tuning a Parallel Segmented Flow Column and Enabling Multiplexed Detection

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

Bioengineering

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented. A major difficulty in adapting PCD to modern HPLC systems and columns is the need for large volume reaction coils that enable reagent mixing and then the derivatization reaction to take place. This large post column dead volume leads to band broadening, which results in a loss of observed separation efficiency and indeed detection in sensitivity. In reaction flow post column derivatization (RF-PCD) the derivatization reagent(s) are pumped against the flow of mobile phase into either one or two of the outer ports of the reaction flow column where it is mixed with column effluent inside a frit housed within the column end fitting. This technique allows for more efficient mixing of the column effluent and derivatization reagent(s) meaning that the volume of the reaction loops can be minimized or even eliminated altogether. It has been found that RF-PCD methods perform better than conventional PCD methods in terms of observed separation efficiency and signal to noise ratio. A further advantage of RF-PCD techniques is the ability to monitor effluent coming from the central port in its underivatized state. RF-PCD has currently been trialed on a relatively small range of post column reactions, however, there is currently no reason to suggest that RF-PCD could not be adapted to any existing one or two component (as long as both reagents are added at the same time) post column derivatization reaction.

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented.

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is ...

Curtain Flow Column: Optimization of Efficiency and Sensitivity

Active Flow Technology (AFT) is a form of column technology that increases the separation performance of a HPLC column through the use of a specially purpose built multiport end-fitting(s). Curtain Flow (CF) columns belong to the AFT suite of columns, specifically the CF column is designed so that the sample is injected into the radial central region of the bed and a curtain flow of mobile phase surrounding the injection of solute prevents the radial dispersion of the sample to the wall. The column functions as an 'infinite diameter' column. The purpose of the design is to overcome the radial heterogeneity of the column bed, and at the same time maximize the sample load into the radial central region of the column bed, which serves to increase detection sensitivity. The protocol described herein outlines the system and CF column set up and the tuning process for an optimized infinite diameter 'virtual' column.

Here, we present a protocol for the operation and optimization of Active Flow Technology (AFT) column in Curtain flow (CF) mode for enhanced separation performance.

Active Flow Technology (AFT) is a form of column technology that increases the separation performance of a HPLC column through the use of a specially ...

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM)

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture &#8211; information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization backend), as well as the workflow of imaging flow cytometry based on ATOM, using human cells and micro-algae as the examples.

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy ATOM

This protocol describes the implementation of an asymmetric-detection time-stretch optical microscopy system for single-cell imaging in ultrafast microfluidic flow and its applications in imaging flow cytometry.

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the ...

Engineering

A Modular Microfluidic Technology for Systematic Studies of Colloidal Semiconductor Nanocrystals

Colloidal semiconductor nanocrystals, known as quantum dots (QDs), are a rapidly growing class of materials in commercial electronics, such as light emitting diodes (LEDs) and photovoltaics (PVs). Among this material group, inorganic/organic perovskites have demonstrated significant improvement and potential towards high-efficiency, low-cost PV fabrication due to their high charge carrier mobilities and lifetimes. Despite the opportunities for perovskite QDs in large-scale PV and LED applications, the lack of fundamental and comprehensive understanding of their growth pathways has inhibited their adaptation within continuous nanomanufacturing strategies. Traditional flask-based screening approaches are generally expensive, labor-intensive, and imprecise for effectively characterizing the broad parameter space and synthesis variety relevant to colloidal QD reactions. In this work, a fully autonomous microfluidic platform is developed to systematically study the large parameter space associated with the colloidal synthesis of nanocrystals in a continuous flow format. Through the application of a novel translating three-port flow cell and modular reactor extension units, the system may rapidly collect fluorescence and absorption spectra across reactor lengths ranging 3 - 196 cm. The adjustable reactor length not only decouples the residence time from the velocity-dependent mass transfer, it also substantially improves the sampling rates and chemical consumption due to the characterization of 40 unique spectra within a single equilibrated system. Sample rates may reach up to 30,000 unique spectra per day, and the conditions cover 4 orders of magnitude in residence times ranging 100 ms - 17 min. Further applications of this system would substantially improve the rate and precision of the material discovery and screening in future studies. Detailed within this report are the system materials and assembly protocols with a general description of the automated sampling software and offline data processing.

Detailed herein are the operation and assembly protocols of a modular microfluidic screening platform for the systematic characterization of colloidal semiconductor nanocrystal syntheses. Through fully adjustable system arrangements, highly efficient spectra collection may be carried out across 4 orders of magnitude reaction time scales within a mass transfer-controlled sampling space.

Colloidal semiconductor nanocrystals, known as quantum dots (QDs), are a rapidly growing class of materials in commercial electronics, such as light ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the averaging that takes place in bulk-phase studies. To achieve visualization, dual optical tweezers, where one trap is fixed and the other is mobile, are focused into one channel of a multi-stream microfluidic chamber positioned on the stage of an inverted fluorescence microscope. The optical tweezers trap single molecules of fluorescently labeled DNA and fluid flow through the chamber and past the trapped beads, stretches the DNA to B-form (under minimal force, i.e., 0 pN) with the nucleic acid being observed as a white string against a black background. DNA molecules are moved from one stream to the next, by translating the stage perpendicular to the flow to enable the initiation of reactions in a controlled manner. To achieve success, microfluidic devices with optically clear channels are mated to glass syringes held in place in a syringe pump. Optimal results use connectors permanently bonded to the flow cell, tubing that is mechanically rigid and chemically resistant, and which is connected to switching valves that eliminate bubbles that prohibit laminar flow.

Visual, single-molecule biochemistry studied through microfluidic chambers is greatly facilitated using glass barrel, gas-tight syringes, stable connections of tubing to flow cells, and elimination of bubbles by placing switching valves between the syringes and tubing. The protocol describes dual optical traps that enable visualization of DNA transactions and intermolecular interactions.