The authors would like to thank the Commission for Technology and Innovation (CTI, Switzerland) for their financial support (No. 13236.1 PFFLI-LS).

The investigations were performed using a piped pump setup (Figure&#160;2), which enables mechanical stress experiments at flow rates up to 60&#160;L&#160;min-1 and pressure drops up to 2&#160;bar to be performed. As shown in Figure&#160;2, the experimental setup is comprised of the storage vessel, the pump circuit, and the equipment for the inline endoscopy technique. The impeller of the storage vessel was only used to mix the surfactant. Peripheral elements were integrated into the closed loop to monitor the flow rate V&#775; and pressure drop p at different pump settings. The investigations were varied by using the hand-wheel valve.
1. Experimental Setup
<ol>
	<li>Ensure that the bioreactor (D&#160;=&#160;0.15&#160;m, H/D&#160;=&#160;2.2) is equipped with an impeller for the dissolution of the surfactant and make sure that the inlet tube dips into the fluid to avoid a gas entering.</li>
	<li>Equip the pump loop with a syringe port, the investigated pump, a clamp-on flow meter, a single-use pressure sensor and a hand-wheel valve. After connection of the pump loop to the storage vessel, connect the pump head to the motor and prepare the endoscope probe.</li>
	<li>Mount the changeable reflection plane, a rhodium mirror in this case, at the probe tip and adjust the distance between mirror and lens to 150&#160;&#181;m. Adjust the screw at 100&#160;&#181;m to focus the sharpness of the objective.</li>
	<li>Connect the probe to the stroboscope via a fiber-optic cable and the camera of the endoscope to the computer via an Ethernet cable. Then, connect the camera and the stroboscope together via a trigger-box cable.</li>
	<li>Start the computer and open the manufacturer-provided software, which includes an image acquisition and recognition software, as well as result analyzer software.</li>
	<li>Select the image acquisition software in the main menu.</li>
	<li>Click on the button &#8220;Detect device&#8221; in the upper left corner of the screen to detect the camera.</li>
	<li>Under &#8220;Directory Settings&#8221; select the location on the computer for saving images and activate the command &#8220;Create trigger sub folders&#8221;.</li>
	<li>Enter the process parameters in section &#8220;Trigger mode: Ready&#8221;. 
	Frame rate: 7.5&#160;Hz 
	Frames per trigger: 50 
	Number of triggers: 60 
	Trigger interval: 60&#160;sec</li>
	<li>After completion of all preparatory work, pour 5&#160;L deionized water into the storage vessel and switch on the pump to fill the pump and the pump loop.</li>
	<li>Switch off the pump and add 0.9 ml of surfactant (csurfactant&#160;=&#160;0.18&#160;ml L-1, &#961;surfactant,20&#176;C &#160;=&#160;1,070&#160;kg&#160;m-3, critical micelle concentration (cmc): &#969;cmc&#160;&#8776;&#160;0.018&#160;ml L-1, &#969;surfactant&#160;&#8776;&#160;10&#160;&#183;&#160;&#969;cmc) with a 10 ml pipette under stirring. After 10&#160;min the surfactant is completely dissolved.</li>
	<li>Switch off the impeller and turn on the pump. Position the endoscope probe so that the lens is located directly beneath the inlet tube.</li>
	<li>Set the flow rate of 3.4&#160;L&#160;min-1 and the pressure drop of 0.03, 0.3 or 0.61&#160;bar by varying the impeller speed and the hand-wheel valve.</li>
	<li>Weigh 6.3&#160;g of oil directly in the syringe (&#946;oil&#160;=&#160;1.26&#160;g&#160;L-1, &#961;oil,20&#176;C&#160;=&#160;989.5&#160;kg&#160;m-3).</li>
	<li>Start the image acquisition software and add the oil via the syringe port. The running pump distributes the emulsion drops.</li>
	<li>After 1 hr, finish the shear stress investigation and clean the inline endoscope as well as the bioreactor with the integrated pump loop. Subsequently, prepare the experimental setup for the next pumping process.</li>
</ol>
2. Measurement and Image Analysis
<ol>
	<li>Open the automatic image recognition software in the main menu.</li>
	<li>Under &#8220;Batch Root Directory&#8221; select the location on the computer for saving the files (all*.csv).</li>
	<li>Select the column &#8220;Image Series Path&#8221; and click on the button &#8220;Add Image Series Subfolders&#8221; in the lower left corner of the screen to load the image series.</li>
	<li>Load the process parameters which are provided by the manufacturer. Select the column &#8220;Search Settings (*.pss or auftrag_*.mat)&#8221; and click on the button &#8220;Set Search Settings&#8221; in the lower-middle of the screen to load process parameters in order to specify the drop recognition.</li>
	<li>Select the column &#8220;Search Pattern (*.psp or F_*.mat) and click on the button &#8220;Set Search Pattern&#8221; in the lower right corner of the screen to load the process parameters in order to specify the drop analysis.</li>
	<li>Start the image recognition by clicking on the button &#8220;Start Batch&#8221;.</li>
	<li>After completion of image recognition, express the detected drop sizes by the Sauter mean diameter (d32), or any other representative mean value or distribution of choice by using the result analyzer software.</li>
	<li>Open the result analyzer software in the main menu.</li>
	<li>Activate the command &#8220;all*.csv in 1 Folder&#8221; and click on the button &#8220;Load Folder(s)&#8221; in the upper left corner of the screen to load the previously saved all*.csv file.</li>
	<li>Select the relevant value (e.g., Sauter mean diameter) in the drop-down list in the upper middle of the screen to visualize the results. For the calculation of the diameter enter the scaling of 0.6591&#160;&#181;m pixel-1 on the right, which is provided by the manufacturer.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/53052/53052fig2.jpg" /> 
Figure 2. Experimental setup. Pump circuit for piped pump setup using inline endoscopy as the measurement technique: (1) storage vessel, (2) syringe port, (3) pump, (4) pressure sensor, (5) flow sensor, (6) stroboscope, (7) computer with the manufacturer-provided software, and (8) endoscope probe. <a href="https://www.jove.com/files/ftp_upload/53052/53052fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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S., 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=Response+of+a+concentrated+monoclonal+antibody+formulation+to+high+shear.">Response of a concentrated monoclonal antibody formulation to high shear.</a> Biotechnol. 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Technol. 23 (7), 579-581 (2000).</li><li>Fries, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Quantifizierung der mechanischen Beanspruchung von Pumpen auf tierische Zellen mittels des nicht-biologischen Modellsystems Emulsion (Quantification of mechanical stress caused by pumps on mammalian cells using a non-biological emulsion model system). , (2014).</li><li>Maa&#223;, S., Wollny, S., Sperling, R., Kraume, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numerical+and+experimental+analysis+of+particle+strain+and+breakage+in+turbulent+dispersions.">Numerical and experimental analysis of particle strain and breakage in turbulent dispersions.</a> Chem. Eng. Res. 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Wolfgang Dornfeld and Reto Sch&#246;b are employees of Levitronix, Ltd., manufacturer of the MagLev centrifugal pumps.

The aim of this protocol is to show the suitability of the inline endoscopy technique for drop size measurements in pumping processes. For this purpose, drop sizes of an oil-water emulsion system were determined and a measured Sauter mean diameter was calculated to characterize the mechanical stress of the MagLev centrifugal pumps as well as their counterparts, a peristaltic and a 4-piston diaphragm pump. The results showed low variation of the measured Sauter mean diameters, which were quantified by standard deviations of below 15%, indicating that drop sizes have been reliably and accurately measured. As a consequence, the measured Sauter mean diameter could successfully be used as a comparison criterion to evaluate the mechanical stress of the pumps investigated. The MagLev centrifugal pumps revealed larger measured Sauter mean diameters, indicating lower mechanical stresses on emulsion drops compared with the peristaltic and 4-piston diaphragm pumps. In studies to date, inline endoscopy has been shown to be a robust and simple technique for reliable drop size measurement1,6,12-14,20-21, which was also confirmed by this study. In comparison to alternative measurement techniques, such as the fiber optical FBR sensor, the FBRM and the 2D-ORM technique, the endoscope technique can be used as the standard method for obtaining precise data in liquid/liquid applications12,14.
The easy handling of the inline endoscopy and the simple production of the non-biological oil-water emulsion system enables a straightforward procedure for drop size detections according to the protocol text (see above). Nevertheless, it should be mentioned that the position of the endoscope probe depends on the fluid flow in the storage vessel. Further investigations (data not shown) have revealed that the lens of the probe should be located directly beneath the inlet tube for lower flow rates up to 5&#160;L&#160;min-1 in order to avoid a multiple detection of one drop19. For sharp images at flow rates over 5&#160;L&#160;min-1, it is recommended to position the probe at least 10&#160;cm away from the inlet tube. Independent of process parameters, the holder of the inline endoscopy should be stable in order to avoid a shifting of the probe, which can result in blurred images.
Furthermore, it should particularly be noted that the drop size detected is close to the lower detection limit of the applied photo-optical system, where the minimum detectable drop diameter is 6.5&#160;&#181;m. As the manufacturer-provided software has been improved, inline endoscopy techniques can reliably detect a minimal drop size of 1&#160;&#181;m. Moreover, the image processing will be further developed to enable online monitoring of industrial applications.
While the present study focused on relatively low flow rates of up to 3.4&#160;L&#160;min-1, future studies should consider a wider range of operation conditions. First investigations have been carried out at flow rates up to 20&#160;L&#160;min-1 (data not shown). However, a 1:2 dilution (csurfactant&#160;=&#160;0.09&#160;ml L-1, coil&#160;=&#160;0.64&#160;ml L-1) of the oil-water emulsion system is recommended at flow rates over 10&#160;L&#160;min-1&#160;19, as increased drop breakup caused by higher mechanical stress would otherwise affect drop detection and reduce the number of drops detected. Tests were carried out with a 1:2 dilution and compared with results of an undiluted oil-water emulsion system. For both approaches, the Sauter mean diameters have been reliably measured (standard deviation below 5%). Therefore, reduced volume fraction (1:2 dilution) did not influence the measured Sauter mean diameters, and thus a drop-drop breakup was negligible.
These powerful experimental approaches provide a good basis for the improvement of the endoscopy technique as well as the related image acquisition, recognition and result analyzer software. Furthermore, the suitability of the endoscopy technique to classify pump types and series according to their mechanical stress was successfully demonstrated. The results obtained are essential for pump design development and the optimization of pumps to reduce cell damage.

Pumps are used to transfer cell cultures in the pharmaceutical and biotechnological industries. During the pumping process, mechanical stress can result in irreversible cell damage, which might impair the quantity and quality of the product1-4. The level of mechanical stress depends on the pump type and pump settings, as demonstrated in previous studies5-6. Commonly, peristaltic, syringe and diaphragm pumps are used for single-use (SU) technology based applications. These pumps result in high local shear forces caused by the compression of the pump tubing and the pulsating flow7.
In order to overcome these drawbacks, magnetically levitated centrifugal pumps (MagLev centrifugal pumps) constitute a promising alternative. The motor is magnetically driven in order to avoid narrow gaps between the impeller and the pump housing (Figure 1). A previous study investigated the MagLev centrifugal pumps and showed lower mechanical stress in Chinese Hamster Ovary (CHO) cells compared with peristaltic and 4-piston diaphragm pumps5. In addition, hemolysis analyses revealed no significant blood trauma and thrombus formation over a range of operation conditions using these pumps8-11. The findings demonstrate that use of these specifically designed pumps applies less mechanical stress on biological systems in comparison with peristaltic and diaphragm pumps. To investigate the mechanical stress with limited experimental expense, an oil-water emulsion model system is recommended due to its cost- (ca. 99.8%) and time-reduced (ca. 99.5%) application compared with biological cell culture systems.
As drop breakup of the oil-water emulsion system is a function of mechanical stress, drop sizes must be counted over the experimental time of shear stress investigations. Many techniques for sizing drops are available, which can be divided into sound, laser and photo based techniques12. In particular, the use of the photo-optical probe inline endoscopy shows almost identical drop sizes for manual and automatic detections (standard deviation below 10%) and enables a detection of 250 drops per minute13. Because of its accuracy and reliability, the endoscope technique has been shown to be an effective standard measurement technique for drop size distributions in liquid/liquid dispersions when compared with other commonly used probes (e.g., fiber optical forward-backward-ratio (FBR) sensor, focused beam reflectance method (FBRM) and the two-dimensional optical reflectance measurement technique (2D-ORM))12,14. Moreover, the suitability of inline endoscopy for measuring drop sizes in a stirred vessel has been demonstrated several times in previous investigations15-18.
Based on a prior study6, this protocol describes the use of inline endoscopy to determine drop sizes (Sauter mean diameter) of an oil-water emulsion system in pumps. The Sauter mean diameter was used as a comparison criterion in order to estimate the mechanical stress of the multi-use (MU) MagLev centrifugal pumps, a peristaltic and a single-use (SU) 4-piston diaphragm pump.
<img alt="Figure 1" src="/files/ftp_upload/53052/53052fig1.jpg" /> 
Figure 1. Magnetically levitated centrifugal pump-system. (A) The principle of a bearingless motor and (B) the PuraLev&#160;200MU are shown as an example. <a href="https://www.jove.com/files/ftp_upload/53052/53052fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>CCD camera</td>
			<td>Allied Vision Technologies GmbH</td>
			<td>GX2750</td>
			<td>Equipment for inline endoscopy</td>
		</tr>
		<tr>
			<td>C-Flex Biopharmaceutical Tubing</td>
			<td>Saint-Gobain Performance Plastics</td>
			<td>374-375-4</td>
			<td>Tube 
			Select a tubing length of about 45 cm 
			before the pump.</td>
		</tr>
		<tr>
			<td>C-Flex Biopharmaceutical Tubing</td>
			<td>Saint-Gobain Performance Plastics</td>
			<td>374-375-3</td>
			<td>Tube 
			Select a tubing length of about 45 cm after the pump and clamp on the flow sensor to this tubing.</td>
		</tr>
		<tr>
			<td>CLAVE Connector</td>
			<td>Victus</td>
			<td>011-C2000</td>
			<td>Sampling port</td>
		</tr>
		<tr>
			<td>Controller LPC-200.1-02</td>
			<td>Levitronix GmbH</td>
			<td>100-30030</td>
			<td>PuraLev 200MU controller</td>
		</tr>
		<tr>
			<td>Controller LPC-600.1-02</td>
			<td>Levitronix GmbH</td>
			<td>100-30033</td>
			<td>PuraLev 600MU controller</td>
		</tr>
		<tr>
			<td>LeviFlow Clamp-On Sensor LFSC-12</td>
			<td>Levitronix GmbH</td>
			<td>100-30329</td>
			<td>Flow sensor for flow rates below 5 L min-1</td>
		</tr>
		<tr>
			<td>LeviFlow Converter LFC-1C-CS</td>
			<td>Levitronix GmbH</td>
			<td>100-30328</td>
			<td>Flow sensor output device&#160;</td>
		</tr>
		<tr>
			<td>Masterflex I/P Easy Load</td>
			<td>Fisher Scientific AG</td>
			<td>EW-77963-10</td>
			<td>Peristaltic pump</td>
		</tr>
		<tr>
			<td>Mitos free flow valve</td>
			<td>Parker Hannifin Europe S&#224;rl</td>
			<td>FFLQR16S6S6AM</td>
			<td>Valve</td>
		</tr>
		<tr>
			<td>Mobil Eal Arctic</td>
			<td>Exxon Mobil Corporation</td>
			<td>Mobil EAL Arctic 22</td>
			<td>Oil 
			Prepare the emulsion directly before 
			the experiment.</td>
		</tr>
		<tr>
			<td>Motor</td>
			<td>Elektromotorenwerk Brienz AG</td>
			<td>7WAC72N4THTF</td>
			<td>Motor for agitator shaft</td>
		</tr>
		<tr>
			<td>Motor BSM-1.4</td>
			<td>Levitronix GmbH</td>
			<td>100-10005</td>
			<td>PuraLev 200MU motor</td>
		</tr>
		<tr>
			<td>Motor LPM-600.4</td>
			<td>Levitronix GmbH</td>
			<td>100-10038</td>
			<td>PuraLev 600MU motor</td>
		</tr>
		<tr>
			<td>Norm-Ject 10 mL Luer Lock</td>
			<td>Restek Corporation</td>
			<td>22775</td>
			<td>Syringe</td>
		</tr>
		<tr>
			<td>Pump Head LPP-200.5</td>
			<td>Levitronix GmbH</td>
			<td>100-90525</td>
			<td>PuraLev 200MU pump head</td>
		</tr>
		<tr>
			<td>Pump Head LPP-600.18</td>
			<td>Levitronix GmbH</td>
			<td>100-90548</td>
			<td>PuraLev 600MU pump head</td>
		</tr>
		<tr>
			<td>Quattroflow 1200-SU</td>
			<td>Almatechnik AG</td>
			<td>QF 1200</td>
			<td>4-piston diaphragm pump</td>
		</tr>
		<tr>
			<td>SciPres Sensor</td>
			<td>SciLog</td>
			<td>080-695PSX</td>
			<td>Pressure sensor</td>
		</tr>
		<tr>
			<td>SciPres Sensor Monitor</td>
			<td>SciLog</td>
			<td>080-690</td>
			<td>Pressure sensor output device&#160;</td>
		</tr>
		<tr>
			<td>SOPAT-VF Inline Endoscopic Probe</td>
			<td>SOPAT GmbH</td>
			<td></td>
			<td>Inline endoscopy</td>
		</tr>
		<tr>
			<td>Stroboscope</td>
			<td>Drello GmbH &#38; Co KG</td>
			<td>Drelloscop 255-01</td>
			<td>Equipment for inline endoscopy</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td>Surfactant 
			Handle with gloves and goggles. 
			(acute toxicity, eye irritation)</td>
		</tr>
	</tbody>
</table>

a cost-effective and reliable method to predict mechanical stress in single-use and standard pumps

Pumps are mainly used when transferring sterile culture broths in biopharmaceutical and biotechnological production processes. However, during the pumping process shear forces occur which can lead to qualitative and/or quantitative product loss. To calculate the mechanical stress with limited experimental expense, an oil-water emulsion system was used, whose suitability was demonstrated for drop size detections in bioreactors1. As drop breakup of the oil-water emulsion system is a function of mechanical stress, drop sizes need to be counted over the experimental time of shear stress investigations. In previous studies, the inline endoscopy has been shown to be an accurate and reliable measurement technique for drop size detections in liquid/liquid dispersions. The aim of this protocol is to show the suitability of the inline endoscopy technique for drop size measurements in pumping processes. In order to express the drop size, the Sauter mean diameter d32 was used as the representative diameter of drops in the oil-water emulsion. The results showed low variation in the Sauter mean diameters, which were quantified by standard deviations of below 15%, indicating the reliability of the measurement technique.

Optical assessment
Figure&#160;3 shows the particle recognition images after a pumping time of 1 hr. The upper four images show the drops before recognition and the lower four images show the drops marked by the recognition software. The drops detected are highlighted with a green edge. Comparing the upper and lower images shows that the drop edges were precisely detected by the image recognition software. The images on the left show the drop distribution for the MagLev centrifugal pumps PuraLev&#160;200MU and PuraLev&#160;600MU, and those on the right show the 4-piston diaphragm and the peristaltic pump. An optical assessment allowed an initial classification of the mechanical stress in the model emulsion system. This revealed that larger drop sizes and lower drop counts were generated by the MagLev centrifugal pumps compared with the 4-piston diaphragm and peristaltic pump. Consequently, the MagLev centrifugal pumps, especially the PuraLev&#160;200MU, showed reduced drop breakage, indicating lower mechanical stresses.
<img alt="Figure 3" src="/files/ftp_upload/53052/53052fig3.jpg" /> 
Figure 3. Images of the inline endoscopy. Emulsion drops before (A, B, C, D) and after (E, F, G, H) particle recognition after 1 hr of pumping using (A, E) the PuraLev&#160;200MU, (B, F) the PuraLev&#160;600MU, (C, G) the 4-piston diaphragm pump, and (D, H) the peristaltic pump under identical operating conditions (3.4&#160;L&#160;min-1 and 0.03&#160;bar). <a href="https://www.jove.com/files/ftp_upload/53052/53052fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Sauter mean diameter
Further investigations revealed standard deviations below d32&#160;&#177;&#160;0.4&#160;&#181;m and guaranteed reproducible results when using inline endoscopy19. Therefore, multiple investigations were not required for this approach, which additionally reduced the experimental expense.
In order to express the drop size, the Sauter mean diameter d32 (see Eq.&#160;1) was used as the representative diameter of drops in the oil-water emulsion for this approach. In general, the Sauter mean diameter decreased over time for all pump types and pump settings until reaching steady state12. Investigations in this study confirmed the progression of the Sauter mean diameter (Figure&#160;4A to D), curves of the PuraLev&#160;200MU (Figure&#160;4A) and the peristaltic pump (Figure&#160;4D) being exemplarily discussed in this protocol.
In contrast to the PuraLev&#160;200MU, the Sauter mean diameters were up to 40% smaller for the peristaltic pump at the same operation conditions (flow rate&#160;=&#160;3.4&#160;L&#160;min-1; pressure drop&#160;=&#160;0.03&#160;bar). As a consequence, higher mechanical stresses resulted in an increased drop breakup and therefore smaller drop sizes. Furthermore, the Sauter mean diameter decreased with increasing pressure drop in the PuraLev&#160;200MU (Figure&#160;4A), which indicated the dependency of drop size on pressure drop. In contrast, the peristaltic pump showed a Sauter mean diameter of d32,60min&#160;=&#160;10&#160;&#181;m at the end of the experiment for all process parameters (Figure&#160;4D). Therefore, the Sauter mean diameter was found to be independent of pressure drop. However, the results reflect physical understanding of the drop breakup: with higher mechanical stress, smaller Sauter mean diameters were determined (see also Figure&#160;5).
For each measurement point, at least 300&#160;drops were determined in order to guarantee statistical certainty. The maximal standard deviation decreased for the PuraLev&#160;200MU from d32,4min&#160;&#177;&#160;42&#160;&#181;m and for the PuraLev&#160;600MU from d32,6min&#160;&#177;&#160;21&#160;&#181;m to approximately d32&#160;&#177;&#160;0.5&#160;&#181;m at the end of the pumping process. The decreased standard deviation resulted from the increased homogeneous drop size distribution until steady state was reached. In comparison to the MagLev centrifugal pumps, the peristaltic and 4-piston diaphragm pumps revealed standard deviations below d32&#160;&#177;&#160;10&#160;&#181;m.
<img alt="Equation 1" src="/files/ftp_upload/53052/53052eq1.jpg" width="80" /> (1)
<img alt="Figure 4" src="/files/ftp_upload/53052/53052fig4.jpg" /> 
Figure 4. Typical profiles of Sauter mean diameters d32 over time and the determination of the measured Sauter mean diameters d32,m. Comparison of Sauter mean diameters d32 (A) for the PuraLev&#160;200MU, (B) for the PuraLev&#160;600MU, (C) for the 4-piston diaphragm pump, and (D) for the peristaltic pump. The Sauter mean diameters d32 were determined at a flow rate of 3.4&#160;L&#160;min-1 and pressure drops ranging from 0.03 to 0.61&#160;bar. The measured Sauter mean diameter d32,m was calculated for the last 10 min&#160;(boundary). The resulting standard deviation of the Sauter mean diameters d32 (N&#160;&#8805;&#160;300) is shown. <a href="https://www.jove.com/files/ftp_upload/53052/53052fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Measured Sauter mean diameter as a comparison system
As described above, the Sauter mean diameter decreased over time until the drop sizes reached steady state. In the last 10&#160;min of the experimental time, the average value of the Sauter mean diameter was calculated to determine the measured Sauter mean diameter, which was used as a comparison criterion (see boundary of Figure&#160;4A-D). The measured Sauter mean diameters d32,m are shown for a flow rate of 3.4&#160;L&#160;min-1 and a pressure drop range from 0.03 to 0.61&#160;bar in Figure&#160;5. Larger measured Sauter mean diameters were determined for both MagLev centrifugal pumps (200MU and 600MU) and the 4-piston diaphragm pump at lower pressure drops and impeller speeds. The peristaltic pump revealed measured Sauter mean diameters of d32,m&#160;=&#160;10&#160;&#181;m for all process parameters. As previously mentioned, shear forces were independent of the pressure drop for the peristaltic pump.
The largest measured Sauter mean diameters of d32,m&#160;=&#160;36&#160;&#181;m for the PuraLev&#160;200MU and d32,m&#160;=&#160;34&#160;&#181;m for the PuraLev&#160;600MU were obtained at a pressure drop of 0.03&#160;bar. In comparison with their counterparts, the MagLev centrifugal pump series obtained up to 59% larger measured Sauter mean diameters. These results indicated a lower rate of drop breakup and thus lower mechanical stress resulting from the use of the centrifugal pumps.
The standard deviation of the measured Sauter mean diameters during the steady state was below 15%, thereby confirming reliable and accurate values for the drop sizes.
<img alt="Figure 5" src="/files/ftp_upload/53052/53052fig5.jpg" /> 
Figure 5. Comparison of measured Sauter mean diameters d32,m. Measured Sauter mean diameters for the MagLev centrifugal pumps and their counterparts at 3.4&#160;L&#160;min-1 and pressure drops of 0.03, 0.30 and 0.61&#160;bar. The resulting standard deviations of the measured Sauter mean diameters d32,m during the steady state are shown.
Abbreviations <a href="https://www.jove.com/files/ftp_upload/53052/53052fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>2D-ORM</td>
			<td>two-dimensional optical reflectance measurement</td>
		</tr>
		<tr>
			<td>CCD</td>
			<td>charge-coupled device</td>
		</tr>
		<tr>
			<td>CHO</td>
			<td>Chinese hamster ovary</td>
		</tr>
		<tr>
			<td>cmc</td>
			<td>critical micelle concentration</td>
		</tr>
		<tr>
			<td>FBR</td>
			<td>forward-backward-ratio</td>
		</tr>
		<tr>
			<td>FBRM</td>
			<td>focused beam reflectance method</td>
		</tr>
		<tr>
			<td>MU</td>
			<td>multi-use</td>
		</tr>
		<tr>
			<td>SU</td>
			<td>single-use</td>
		</tr>
	</tbody>
</table>
Nomenclature
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td><img alt="V" fo:content-="" src="/files/ftp_upload/53052/V.jpg" width="8
" /></td>
			<td>[m3 sec-1]</td>
			<td>flow rate</td>
		</tr>
		<tr>
			<td>c</td>
			<td>[m3 m-3]</td>
			<td>concentration</td>
		</tr>
		<tr>
			<td>d32</td>
			<td>[m]</td>
			<td>Sauter mean diameter</td>
		</tr>
		<tr>
			<td>d32,m</td>
			<td>[m]</td>
			<td>measured Sauter mean diameter</td>
		</tr>
		<tr>
			<td>ds</td>
			<td>[m]</td>
			<td>surface diameter</td>
		</tr>
		<tr>
			<td>dv</td>
			<td>[m]</td>
			<td>volume diameter</td>
		</tr>
		<tr>
			<td>f</td>
			<td>[Hz]</td>
			<td>frequency</td>
		</tr>
		<tr>
			<td>n</td>
			<td>[sec-1]</td>
			<td>impeller speed</td>
		</tr>
		<tr>
			<td>N</td>
			<td>[-]</td>
			<td>number of drops</td>
		</tr>
		<tr>
			<td>p</td>
			<td>Pa</td>
			<td>pressure drop</td>
		</tr>
		<tr>
			<td>t</td>
			<td>sec</td>
			<td>time</td>
		</tr>
		<tr>
			<td>&#946;</td>
			<td>[kg m-3]</td>
			<td>mass concentration</td>
		</tr>
		<tr>
			<td>&#961;</td>
			<td>[kg m-3]</td>
			<td>density</td>
		</tr>
		<tr>
			<td>&#969;</td>
			<td>[m3 m-3]</td>
			<td>mass fraction</td>
		</tr>
	</tbody>
</table>
Table 1. Table of abbreviations and nomenclature.

Watch this Scientific Journal Video about A Cost-effective and Reliable Method to Predict Mechanical Stress in Single-use and Standard Pumps at JoVE.com

A Cost-effective and Reliable Method to Predict Mechanical Stress in Single-use and Standard Pumps

Shear stress investigations on an oil-water emulsion system result in drop breakup over the experimental time. To count drop sizes in pumping processes, the suitability of inline endoscopy was successfully demonstrated in this protocol.

Pumps are mainly used when transferring sterile culture broths in biopharmaceutical and biotechnological production processes. However, during the pumping ...

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9.3K Views.  Zurich University of Applied Sciences. Shear stress investigations on an oil-water emulsion system result in drop breakup over the experimental time. To count drop sizes in pumping processes, the suitability of inline endoscopy was successfully demonstrated in this protocol. 

Video: A Cost-effective and Reliable Method to Predict Mechanical Stress in Single-use and Standard Pumps

A Method to Fabricate Disconnected Silver Nanostructures in 3D

The standard nanofabrication toolkit includes techniques primarily aimed at creating 2D patterns in dielectric media. Creating metal patterns on a submicron scale requires a combination of nanofabrication tools and several material processing steps. For example, steps to create planar metal structures using ultraviolet photolithography and electron-beam lithography can include sample exposure, sample development, metal deposition, and metal liftoff. To create 3D metal structures, the sequence is repeated multiple times. The complexity and difficulty of stacking and aligning multiple layers limits practical implementations of 3D metal structuring using standard nanofabrication tools. Femtosecond-laser direct-writing has emerged as a pre-eminent technique for 3D nanofabrication.1,2 Femtosecond lasers are frequently used to create 3D patterns in polymers and glasses.3-7 However, 3D metal direct-writing remains a challenge. Here, we describe a method to fabricate silver nanostructures embedded inside a polymer matrix using a femtosecond laser centered at 800 nm. The method enables the fabrication of patterns not feasible using other techniques, such as 3D arrays of disconnected silver voxels.8 Disconnected 3D metal patterns are useful for metamaterials where unit cells are not in contact with each other,9 such as coupled metal dot10,11or coupled metal rod12,13 resonators. Potential applications include negative index metamaterials, invisibility cloaks, and perfect lenses.
In femtosecond-laser direct-writing, the laser wavelength is chosen such that photons are not linearly absorbed in the target medium. When the laser pulse duration is compressed to the femtosecond time scale and the radiation is tightly focused inside the target, the extremely high intensity induces nonlinear absorption. Multiple photons are absorbed simultaneously to cause electronic transitions that lead to material modification within the focused region. Using this approach, one can form structures in the bulk of a material rather than on its surface.
Most work on 3D direct metal writing has focused on creating self-supported metal structures.14-16 The method described here yields sub-micrometer silver structures that do not need to be self-supported because they are embedded inside a matrix. A doped polymer matrix is prepared using a mixture of silver nitrate (AgNO3), polyvinylpyrrolidone (PVP) and water (H2O). Samples are then patterned by irradiation with an 11-MHz femtosecond laser producing 50-fs pulses. During irradiation, photoreduction of silver ions is induced through nonlinear absorption, creating an aggregate of silver nanoparticles in the focal region. Using this approach we create silver patterns embedded in a doped PVP matrix. Adding 3D translation of the sample extends the patterning to three dimensions.

Femtosecond-laser direct-writing is frequently used to create three-dimensional (3D) patterns in polymers and glasses. However, patterning metals in 3D remains a challenge. We describe a method for fabricating silver nanostructures embedded inside a polymer matrix using a femtosecond laser centered at 800 nm.

The standard nanofabrication toolkit includes  techniques primarily aimed at creating 2D patterns in dielectric media. Creating  metal patterns on a ...

Micro 3D Printing Using a Digital Projector and its Application in the Study of Soft Materials Mechanics

Buckling is a classical topic in mechanics. While buckling has long been studied as one of the major structural failure modes1, it has recently drawn new attention as a unique mechanism for pattern transformation. Nature is full of such examples where a wealth of exotic patterns are formed through mechanical instability2-5. Inspired by this elegant mechanism, many studies have demonstrated creation and transformation of patterns using soft materials such as elastomers and hydrogels6-11. Swelling gels are of particular interest because they can spontaneously trigger mechanical instability to create various patterns without the need of external force6-10. Recently, we have reported demonstration of full control over buckling pattern of micro-scaled tubular gels using projection micro-stereolithography (P&mu;SL), a three-dimensional (3D) manufacturing technology capable of rapidly converting computer generated 3D models into physical objects at high resolution12,13. Here we present a simple method to build up a simplified P&mu;SL system using a commercially available digital data projector to study swelling-induced buckling instability for controlled pattern transformation.
A simple desktop 3D printer is built using an off-the-shelf digital data projector and simple optical components such as a convex lens and a mirror14. Cross-sectional images extracted from a 3D solid model is projected on the photosensitive resin surface in sequence, polymerizing liquid resin into a desired 3D solid structure in a layer-by-layer fashion. Even with this simple configuration and easy process, arbitrary 3D objects can be readily fabricated with sub-100 &mu;m resolution.
This desktop 3D printer holds potential in the study of soft material mechanics by offering a great opportunity to explore various 3D geometries. We use this system to fabricate tubular shaped hydrogel structure with different dimensions. Fixed on the bottom to the substrate, the tubular gel develops inhomogeneous stress during swelling, which gives rise to buckling instability. Various wavy patterns appear along the circumference of the tube when the gel structures undergo buckling. Experiment shows that circumferential buckling of desired mode can be created in a controlled manner. Pattern transformation of three-dimensionally structured tubular gels has significant implication not only in mechanics and material science, but also in many other emerging fields such as tunable matamaterials.

We demonstrate controlled pattern transformation of swelling gel tubes by elastic instability. A simple projection micro stereo-lithography setup is built using an off-the-shelf digital data projector to fabricate three-dimensional polymeric structures in a layer-by-layer fashion. Swelling hydrogel tubes under mechanical constraint display various circumferential buckling modes depending on dimension.

Buckling is a classical topic in mechanics. While buckling has long been studied as one of the major structural failure modes1, it has recently drawn new ...

Mechanical Expansion of Steel Tubing as a Solution to Leaky Wellbores

Wellbore cement, a procedural component of wellbore completion operations, primarily provides zonal isolation and mechanical support of the metal pipe (casing), and protects metal components from corrosive fluids. These are essential for uncompromised wellbore integrity. Cements can undergo multiple forms of failure, such as debonding at the cement/rock and cement/metal interfaces, fracturing, and defects within the cement matrix. Failures and defects within the cement will ultimately lead to fluid migration, resulting in inter-zonal fluid migration and premature well abandonment. Currently, there are over 1.8 million operating wells worldwide and over one third of these wells have leak related problems defined as Sustained Casing Pressure (SCP)1. 
The focus of this research was to develop an experimental setup at bench-scale to explore the effect of mechanical manipulation of wellbore casing-cement composite samples as a potential technology for the remediation of gas leaks. 
The experimental methodology utilized in this study enabled formation of an impermeable seal at the pipe/cement interface in a simulated wellbore system. Successful nitrogen gas flow-through measurements demonstrated that an existing microannulus was sealed at laboratory experimental conditions and fluid flow prevented by mechanical manipulation of the metal/cement composite sample. Furthermore, this methodology can be applied not only for the remediation of leaky wellbores, but also in plugging and abandonment procedures as well as wellbore completions technology, and potentially preventing negative impacts of wellbores on subsurface and surface environments.

This article reports on a laboratory scale investigation of an existing field procedure and its adaptation for sealing of leaky wellbores. It consists of mechanical expansion of metal pipe, which results in an improved metal/cement bond, ultimate sealing of hydraulic pathways and prevention of gas leaks caused by the presence of a microannular channel.

Wellbore cement, a procedural component of wellbore completion operations, primarily provides zonal isolation and mechanical support of the metal pipe ...

A Robotic Platform to Study the Foreflipper of the California Sea Lion

The California sea lion (Zalophus californianus), is an agile and powerful swimmer. Unlike many successful swimmers (dolphins, tuna), they generate most of their thrust with their large foreflippers. This protocol describes a robotic platform designed to study the hydrodynamic performance of the swimming California sea lion (Zalophus californianus). The robot is a model of the animal&#39;s foreflipper that is actuated by motors to replicate the motion of its propulsive stroke (the &#39;clap&#39;). The kinematics of the sea lion&#39;s propulsive stroke are extracted from video data of unmarked, non-research sea lions at the Smithsonian Zoological Park (SNZ). Those data form the basis of the actuation motion of the robotic flipper presented here. The geometry of the robotic flipper is based a on high-resolution laser scan of a foreflipper of an adult female sea lion, scaled to about 60% of the full-scale flipper. The articulated model has three joints, mimicking the elbow, wrist and knuckle joint of the sea lion foreflipper. The robotic platform matches dynamics properties&#8212;Reynolds number and tip speed&#8212;of the animal when accelerating from rest. The robotic flipper can be used to determine the performance (forces and torques) and resulting flowfields.

A robotic platform is described that will be used to study the hydrodynamic performance&#8212;forces and flowfields&#8212;of the swimming California sea lion. The robot is a model of the animal&#39;s foreflipper that is actuated by motors to replicate the motion of its propulsive stroke (the &#39;clap&#39;).

The California sea lion (Zalophus californianus), is an agile and powerful swimmer. Unlike many successful swimmers (dolphins, tuna), they generate most ...

Ice Generation and the Heat and Mass Transfer Phenomena of Introducing Water to a Cold Bath of Brine

We demonstrate a method for the study of the heat and mass transfer and of the freezing phenomena in a subcooled brine environment. Our experiment showed that, under the proper conditions, ice can be produced when water is introduced to a bath of cold brine. To make ice form, in addition to having the brine and water mix, the rate of heat transfer must bypass that of mass transfer. When water is introduced in the form of tiny droplets to the brine surface, the mode of heat and mass transfer is by diffusion. The buoyancy stops water from mixing with the brine underneath, but as the ice grows thicker, it slows down the rate of heat transfer, making ice more difficult to grow as a result. When water is introduced inside the brine in the form of a flow, a number of factors are found to influence how much ice can form. Brine temperature and concentration, which are the driving forces of heat and mass transfer, respectively, can affect the water-to-ice conversion ratio; lower bath temperatures and brine concentrations encourage more ice to form. The flow rheology, which can directly affect both the heat and mass transfer coefficients, is also a key factor. In addition, the flow rheology changes the area of contact of the flow with the bulk fluid.

Here, we present a protocol to demonstrate the generation of ice when water is introduced to a cold bath of brine, as a secondary refrigerant, at a range of temperatures well below the freezing point of water. It can be used as an alternative way of producing ice for industry.

We demonstrate a method for the study of the heat and mass transfer and of the freezing phenomena in a subcooled brine environment. Our experiment showed ...

Plasmonic Trapping and Release of Nanoparticles in a Monitoring Environment

Plasmonic tweezers use surface plasmon polaritons to confine polarizable nanoscale objects. Among the various designs of plasmonic tweezers, only a few can observe immobilized particles. Moreover, a limited number of studies have experimentally measured the exertable forces on the particles. The designs can be classified as the protruding nanodisk type or the suppressed nanohole type. For the latter, microscopic observation is extremely challenging. In this paper, a new plasmonic tweezer system is introduced to monitor particles, both in directions parallel and orthogonal to the symmetric axis of a plasmonic nanohole structure. This feature enables us to observe the movement of each particle near the rim of the nanohole. Furthermore, we can quantitatively estimate the maximal trapping forces using a new fluidic channel.

A microchip fabrication process that incorporates plasmonic tweezers is presented here. The microchip enables the imaging of a trapped particle to measure maximal trapping forces.

Plasmonic tweezers use surface plasmon polaritons to confine polarizable nanoscale objects. Among the various designs of plasmonic tweezers, only a few ...

Experimental Implementation of a New Composite Fabrication Method: Exposing Bare Fibers on the Composite Surface by the Soft Layer Method

The bipolar plate is a key component in proton exchange membrane fuel cells (PEMFCs) and vanadium redox flow batteries (VRFBs). It is a multi-functional component that should have high electrical conductivity, high mechanical properties, and high productivity.
In this regard, a carbon fiber/epoxy resin composite can be an ideal material to replace the conventional graphite bipolar plate, which often leads to the catastrophic failure of the entire system because of its inherent brittleness. Though the carbon/epoxy composite has high mechanical properties and is easy to manufacture, the electrical conductivity in the through-thickness direction is poor because of the resin-rich layer that forms on its surface. Therefore, an expanded graphite coating was adopted to solve the electrical conductivity issue. However, the expanded graphite coating not only increases the manufacturing costs but also has poor mechanical properties.
In this study, a method to expose fibers on the composite surface is demonstrated. There are currently many methods that can expose fibers by surface treatment after the fabrication of the composite. This new method, however, does not require surface treatment because the fibers are exposed during the manufacture of the composite. By exposing bare carbon fibers on the surface, the electrical conductivity and mechanical strength of the composite are increased drastically.

A protocol to expose bare fibers on the composite surface by eliminating resin rich area is presented. The fibers are exposed during fabrication of the composites, not by the post surface treatment. The exposed carbon composites exhibit high electrical conductivity in the through-thickness direction and high mechanical property.

The bipolar plate is a key component in proton exchange membrane fuel cells (PEMFCs) and vanadium redox flow batteries (VRFBs). It is a multi-functional ...

Measurements of Waves in a Wind-wave Tank Under Steady and Time-varying Wind Forcing

This manuscript describes an experimental procedure that allows obtaining diverse quantitative information on temporal and spatial evolution of water waves excited by time-dependent and steady wind forcing. Capacitance-type wave gauge and Laser Slope Gauge (LSG) are used to measure instantaneous water surface elevation and two components of the instantaneous surface slope at a number of locations along the test section of a wind-wave facility. The computer-controlled blower provides airflow over the water in the tank whose rate can vary in time. In the present experiments, the wind speed in the test section initially increases quickly from rest to the set value. It is then kept constant for the prescribed duration; finally, the airflow is shut down. At the beginning of each experimental run, the water surface is calm and there is no wind. Operation of the blower is initiated simultaneously with the acquisition of data provided by all sensors by a computer; data acquisition continues until the waves in the tank fully decay. Multiple independent runs performed under identical forcing conditions allow determining statistically reliable ensemble-averaged characteristic parameters that quantitatively describe wind-waves&#39; variation in time for the initial development stage as a function of fetch. The procedure also allows characterizing the spatial evolution of the wave field under steady wind forcing, as well as decay of waves in time, once the wind is shut down, as a function of fetch.

This manuscript describes a fully computer-controlled procedure that allows obtaining reliable statistical parameters from experiments of water waves excited by steady and unsteady wind forcing in a small-scale facility.

This manuscript describes an experimental procedure that allows obtaining diverse quantitative information on temporal and spatial evolution of water ...

Measurements of Local Instantaneous Convective Heat Transfer in a Pipe - Single and Two-phase Flow

This manuscript provides step by step description of the manufacturing process of a test section designed to measure the local instantaneous heat transfer coefficient as a function of the liquid flow rate in a transparent pipe. With certain amendments, the approach is extended to gas-liquid flows, with a particular emphasis on the effect of a single elongated (Taylor) air bubble on heat transfer enhancement. A non-invasive thermography technique is applied to measure the instantaneous temperature of a thin metal foil heated electrically. The foil is glued to cover a narrow slot cut in the pipe. The thermal inertia of the foil is small enough to detect the variation in the instantaneous foil temperature. The test section can be moved along the pipe and is long enough to cover a considerable part of the growing thermal boundary layer.
At the beginning of each experimental run, a steady state with a constant water flow rate and heat flux to the foil is attained and serves as the reference. The Taylor bubble is then injected into the pipe. The heat transfer coefficient variations due to the passage of a Taylor bubble propagating in a vertical pipe is measured as function of the distance of the measuring point from the bottom of the moving Taylor bubble. Thus, the results represent the local heat transfer coefficients. Multiple independent runs preformed under identical conditions allow accumulating sufficient data to calculate reliable ensemble-averaged results on the transient convective heat transfer. In order to perform this in a frame of reference moving with the bubble, the location of the bubble along the pipe has to be known at all times. Detailed description of measurements of the length and of the translational velocity of the Taylor bubbles by optical probes is presented.

This manuscript describes methods aimed at measuring the local instantaneous convective heat transfer coefficients in a single or two-phase pipe flow. A simple optical method to determine the length and the propagation velocity of an elongated (Taylor) air bubble moving at a constant velocity is also presented.

This manuscript provides step by step description of the manufacturing process of a test section designed to measure the local instantaneous heat transfer ...

Three-electrode Coin Cell Preparation and Electrodeposition Analytics for Lithium-ion Batteries

As lithium-ion batteries find use in high energy and power applications, such as in electric and hybrid-electric vehicles, monitoring the degradation and subsequent safety issues becomes increasingly important. In a Li-ion cell setup, the voltage measurement across the positive and negative terminals inherently includes the effect of the cathode and anode which are coupled and sum to the total cell performance. Accordingly, the ability to monitor the degradation aspects associated with a specific electrode is extremely difficult because the electrodes are fundamentally coupled. A three-electrode setup can overcome this problem. By introducing a third (reference) electrode, the influence of each electrode can be decoupled, and the electrochemical properties can be measured independently. The reference electrode (RE) must have a stable potential that can then be calibrated against a known reference, for example, lithium metal. The three-electrode cell can be used to run electrochemical tests such as cycling, cyclic voltammetry, and electrochemical impedance spectroscopy (EIS). Three-electrode cell EIS measurements can elucidate the contribution of individual electrode impedance to the full cell. In addition, monitoring the anode potential allows the detection of electrodeposition due to lithium plating, which can cause safety concerns. This is especially important for the fast charging of Li-ion batteries in electric vehicles. In order to monitor and characterize the safety and degradation aspects of an electrochemical cell, a three-electrode setup can prove invaluable. This paper aims to provide a guide to constructing a three-electrode coin cell setup using the 2032-coin cell architecture, which is easy to produce, reliable, and cost-effective.

Three-electrode cells are useful in studying the electrochemistry of lithium-ion batteries. Such an electrochemical setup allows the phenomena associated with the cathode and anode to be decoupled and examined independently. Here, we present a guide for construction and use of a three-electrode coin cell with emphasis on lithium plating analytics.

As lithium-ion batteries find use in high energy and power applications, such as in electric and hybrid-electric vehicles, monitoring the degradation and ...