jks2012june - level a project report (3)

James Keaton Smith

Level A Project Report

September 2012

Automation of Chitosan Sponge Neutralization

James Keaton Smith Level A Project Report

Introduction

There are two purposes for this investigation. (1) In May of 2011, I conditionally passed

the University of Memphis Department of Biomedical Engineering Level A exam. The condition

was that I successfully complete an instrumentation related project as defined by my major

advisor and the Level A exam committee. This report will detail the project results to meet this

requirement. (2) In the process of manufacturing a biomaterial device currently under

investigation, the chitosan sponge, there is a neutralization step that is manually performed to

remove residual acids using a sodium hydroxide solution. This project will automate the

neutralization procedure, reducing human error to produce chitosan sponges with a consistently

neutral pH.

Table of Contents:

Title Page 1

Introduction 2

Table of Contents 2

List of Figures 3

Background 4

Materials and Methods 5

Neutralizer Prototype Setup 5

Neutralizer Circuitry Setup 8

Neutralizer Controller Setup 10

Results and Discussion 12

Conclusion 17

References 18

Appendix A: pH Electrode

Appendix B: NI ELVIS II+

Appendix C: NI myDAQ

Appendix D: Half-H Driver


List of Figures

Figure 1: Automatic Neutralizer Design Layout 6

Figure 2: Automatic Neutralizer Block Diagram 6

Figure 3: Automatic Neutralizer Setup 7

Figure 4: Circuit Diagram 9

Figure 5: Automatic Neutralizer Circuitry Setup 9

Figure 6: LabVIEW Block Diagram 11

Figure 7: LabVIEW Virtual Instrument 12

Figure 8: pH Electrode Sensitivity (With pH Meter) 13

Figure 9: pH Electrode Sensitivity (With Designed Project) 13

Figure 10: Neutralizer Voltage Signal Obtained using Standard Buffer Solutions 14

Figure 11: Neutralizer Voltage Signal Obtained using a Chitosan Sponge 15

Figure 12: Chitosan Sponge SEM 15

Figure 13: Improved Design Layout 16


Background

Chitosan is a naturally occurring, linear polysaccharide composed of randomly

distributed β-(1-4)-2-amino-2-D-glucosamine (deacetylated) and β-(1-4)-2-acetamido-2-D-

glucoseamine (acetylated) units.1-3

Chitosan is a cationic weak base, insoluble in water and

organic solvents, but soluble in dilute acid solutions such as acetic, citric, propionic, ascorbic,

lactic, glycolic and other organic and inorganic acids.2,4

Chitosan’s unique charge gives it

bioadhesive properties that allow it to bind to negatively charged surfaces, such as biological

tissues present at a site of trauma or negatively charged implanted devices.5 Chitosan is known to

be biodegradable, antibacterial and able to store and release drugs over time. Applications for the

use of chitosan are estimated to be over 200.1 These applications include cosmetics, agriculture,

food, biomedical, and textile industries. The chitosan sponge is a device currently being

investigated for use in wound dressings, orthopedic implant coatings, drug delivery devices,

infection therapy and other applications.3,4,6

Many factors, such as the degree of deacetylatation,

acid solvent, and manufacturing method, all affect how this chitosan sponge may perform

clinically. Acidic forms of chitosan have been shown to reduce bacterial contamination in

wounds and increase the chitosan device’s degradation, however introducing such a concentrated

acid to a wound site may inhibit wound healing. The neutralization of the chitosan sponge device

minimizes detrimental effects to wound healing from this source, and allows for the sponge’s

controlled degradation.

The standard method for manufacturing a chitosan sponge is to dissolve 5.0 g of 71%

deacetylated chitosan (Primex, Iceland) in 500.0 ml of 1% (v/v) blended lactic/acetic acid (Fisher

Scientific, Pittsburg, PA) solvent (75:25 ratio, respectively). The chitosan solution is stirred for

approximately 5 hours and then filtered through a 180 µm nylon mesh (Gilson, Lewis Center,

OH) to remove insoluble particulates. After filtering, the chitosan is cast into a plastic container

at approximately 7 ml/cm2 and frozen at ‒80°C. The frozen chitosan is placed into a lyophilizer

(FreeZone 2.5; Labconco, Kansas City, MO) until the sample is dehydrated. The dehydrated,

acidic chitosan sponge is then neutralized by submerging it in a 1M sodium hydroxide (NaOH;

Fisher Scientific, Pittsburgh, PA) solution for approximately 10 minutes with constant manual

compression and agitation. The sponge is then physically compressed to remove most of the

basic solution, and this procedure is repeated at least 4 times using distilled/deionized water. At

this point, the sponge is rehydrated in a minimal volume of water and the resulting pH is

measured using a pH electrode and meter (Accumet; Fisher Scientific, Waltham, MA). If the pH

is not > 6.5 and < 7.5, then the sponge is rinsed with water additional times. After the sponge is

neutralized, it is then once again frozen at ‒80°C and lyophilized, to yield the finalized

neutralized chitosan sponge device.

This method of neutralization relies heavily on the manufacturer’s proficiency and

diligence to create chitosan sponges that are consistently at neutral pH, throughout the entire

sponge. Deficiencies in this procedure can produce large sponges with acidic areas or replicate

sponges each with varied pH. This project will provide one possible, non-optimized sponge

neutralization method that reduces human error by automating the procedure using a pH

feedback control system.


Materials and Methods

The first step is to define the physical system to be controlled and what specifications are

needed based on the neutralization procedure requirements. In order to neutralize a chitosan

sponge, standard practice is to wash the sponge with a basic solution, forming a salt and water

from the acid and base combination. This process does not guarantee that the resulting solution is

neutral. When the weak acids in the chitosan sponge are neutralized with a strong base (sodium

hydroxide), the reaction yields water, a sodium spectator ion, and the conjugate base of the weak

acid used. To aid in removing residual acids, bases, or salts formed in the neutralization reaction

the resulting solution and sponge is washed with copious amounts of deionized water (whose pH

is slightly below 7 due to carbonic acid formation).

Neutralizer Prototype Setup

The neutralization chamber was created by cutting a 1” diameter hole into the sidewall of

a 1L high-density polyethylene container (Nalgene; Fisher Scientific, Waltham, MA). The

threaded end of a polypropylene single-barbed tube fitting adapter for a 1” tube inner diameter to

1” national pipe tube male pipe (McMaster-Carr, Aurora, OH) was inserted into the polyethylene

container’s hole. The adaptor’s threaded end was sealed to the container’s wall using a liquid

rubber coating (Flexible Sealer; Home Armor, Memphis, TN). A 3’ long santoprene

rubber/plastic tube with 1” inner diameter (McMaster-Carr, Aurora, OH) was connected to the

adaptor. This 1” inner diameter tube was used as an overflow/waste line, leading from the

neutralization chamber to a nearby sink. When running, the neutralizer’s overflow/waste was

diluted with tap water from the sink’s faucet before draining.

The neutralization chamber was placed on the laboratory bench while two 20L high

density polyethylene containers (Encore Plastics Corp., Byesville, OH) were placed on a mobile

cart, next to the laboratory bench. These 20L containers were used as reservoirs for either

distilled water or 1M sodium hydroxide (Fisher Scientific, Pittsburg, PA) solutions. One

3.8L/min mini DC, brushless, submersible water pump (LightObject; Annex Depot Inc., Elk

Grove, CA) was placed in each of the 20L reservoir containers. These pumps required a 12V at

500mA input and are rated to work in a solution with a pH of 5 to 10. The pumps have a built-in

solution intake filter and an outlet 7.2mm outer diameter outlet. Approximately 4’ long, clear

PVC tubing with a 7mm inner diameter (McMaster-Carr) was connected to the outlet of each

solution reservoir pump. The pump tubing was led to the neutralization chamber and held in

place using a three pronged clamp connected to a cast-iron tripod-base support (Fisher

Scientific).

An Accumet, liquid-filled mercury-free pH combination electrode (Fisher Scientific) was

positioned to where the body of the electrode was submersed in the neutralization chamber

solution and held in place by an electrode holder (Fisher Scientific). The pH electrode’s BNC

connection was converted using an adapter to a 3.5mm mono cable (Radio Shack, Fort Worth,

TX). Figure 1 shows the design layout for the system previously described, figure 2 indicates the

functional block diagram and figure 3 shows the actual neutralizer setup.


Figure 1: Automatic Neutralizer Design Layout

Figure 2: Automatic Neutralizer Block Diagram


Figure 3: Automatic Neutralizer Setup


Neutralizer Circuitry Setup

One NI ELVIS II+ prototyping platform (National Instruments, Austin, TX) and one NI

myDAQ portable measurement and control device (National Instruments) with a Protoboard kit

accessory (Elenco, Wheeling, IL) were connected via USB to a laptop computer equipped with

NI LabVIEW 2012 software (National Instruments).

The pH electrode’s converted 3.5mm mono cable was connected to the 16 bit ADC

resolution analog input of the NI ELVIS II+ prototyping platform. The electrode functions for a

pH range from 0 to 14. The slope of the electrode, out of the box, is 59±3mV/pH unit at 25°C.

The electrode is reported to have a less than 3mV drift per 24 hours in 7 pH buffer (see

Appendix A).

The NI ELVIS II+ and the NI myDAQ were each only able to control and power one

pump each. The NI ELVIS II+ had 8 different analog input channels and an ADC resolution of

16 bits. The NI ELVIS II+ analog inputs had a maximum sampling rate of 1.25MS/s in single

channel and 1.00MS/s in multi-channel with a timing resolution of 50ns. The analog input

impedance when the device was on was greater than 1×1010

Ω. This high input impedance is

necessary because the resistance of the glass membrane pH electrode is exceptionally high, at

300MΩ, although the exact resistance was not found for this specific sensor. There are 2

channels for analog output on the NI ELVIS II+ with a 16 bits DAC resolution with a maximum

update rate of 2.8MS/s for 1 channel with a timing resolution of 50ns. The NI ELVIS II+ has an

output range of ±10V (see Appendix B for full specifications). The NI ELVIS II+ was powered

by an AC/DC converter power supply whereas the NI myDAQ was powered by the laptop

computer via the USB cable connection. The NI myDAQ contained two analog output channels

configured for voltage output. NI myDAQ could generate up to ±10V output signals updated at

up to 200kS/s per channel. There were three power supplies available for use on the NI myDAQ

(see Appendix C for full specifications). The analog pump outputs from the prototyping boards

were connected to a quadruple half-H motor driver (L293DNE; Texas Instruments, Dallas, TX;

see Appendix D for full specifications). The motor driver could provide bidirectional currents of

up to 600mA at voltages from 4.5V to 36V. Each output is a complete totem-pole drive circuit,

enabled in pairs, with drivers 1 and 2 enabled by 1,2EN and drivers 3 and 4 enabled by 3,4EN.

When the enable input is high, the associated drivers are enabled, and their outputs are active.

When the enable input is low, those drivers are disabled and their outputs are off. When

connected with the proper inputs, each pair of drivers can form a reversible drive, but was not

used in this project.

The driver was soldered to a perfboard along with copper wire extensions of the

connections used to provide a more permanent circuit. The NI myDAQ +15V power supply was

connected to the VCC1 terminal, position 16, and grounded. The NImyDAQ, analog output

channel 0, Vin1, was connected to the 1,2EN terminal, at position 1, and grounded. The first

pump was grounded and connected to the driver 1 output at position 3. This pump was placed in

the 20L, water reservoir container. The NI ELVIS II+ +15V power supply was connected to the

VCC2 terminal, position 8, and grounded. The NI ELVIS II+, analog output channel 0, Vin2, was

connected to the 3,4EN terminal, at position 9, and grounded. The second pump was grounded

and connected to the driver 4 output at position 14. This pump was placed in the 20L, base

reservoir container. The 15V of direct current power supply turned the pumps either on or off,

controlled by the software. The heat sinks and ground terminals at positions 4, 5, 12, and 13 were

all grounded. Figure 4 depicts this circuit while figure 5 shows the actual setup.


Figure 4: Circuit Diagram

Figure 5: Automatic Neutralizer Circuitry Setup


Neutralizer Controller Setup

In order to automate the sponge neutralization, it is necessary to control the pH of the

sponge solution. A common method of pH measurement, via a pH glass electrode (Accumet by

Fisher Scientific; Waltham, MA), was used to measure the neutralizing solution’s pH. This form

of ion-selective electrode measures the voltage produced between a measurement electrode and a

reference electrode. This voltage may be correlated to the concentration of hydronium ions in a

solution, and applied in a control system to pump either a base or neutral solution, used to wash

the chitosan sponge. The resulting pH of the chitosan sponge may be continually monitored and

either the basic or neutral solution applied accordingly. If the pH of the resulting solution was

acidic, the base solution pump was switched on until the resulting solution pH is at or above pH

6.5. Then, the water reservoir pump is switched on and remains on unless the pH dips below 6.5,

or until the operator turns off the system. The water pumps were connected to a quadruple half-H

driver used to regulate the current flowing through the circuit as shown in figure 5. Figure 5

shows the circuit connections leading from the data acquisition (DAQ) controller to the two

pumps. Each pump was controlled by an individual DAQ system due to neither DAQ being

capable of controlling both pumps simultaneously.

NI Labview 2012 Student Edition was used to build a virtual instrument that would

receive the pH signal input from the pH sensor and control the outputs (simply on or off

depending on the pH) to the pumps (figures 4 and 5). Voltage inputs were compared to known

pH buffered solutions and analyzed by linear regression in to determine the electrode voltage

resulting from a pH 6.5 solution (figure 8). The software setup is shown in figures 6 and 7. As

the programming of the LabVIEW control system and virtual instrument required the knowledge

to use the software, aid from fellow graduate students well versed in LabVIEW programming

was enlisted. The software was programmed to record the measured signal with a 100Hz sample

rate.


Figure 6: LabVIEW Block Diagram for Feedback Control


Figure 7: LabVIEW Virtual Instrument for Feedback Control

Results and Discussion

This pH control system directs the flow of either a basic or neutral solution determined

by the continually measured pH input from the neutralization container. The potentiometric

method of measuring pH is based on the Nernst equation which describes the relationship

between the electrode’s galvanic potential and the chemical activity of the ion concentration

being measured. The reversal potential of one pH unit change at 25°C for the electrode is

59.16mV. The potential change is inversely related to the pH change, where a decrease in one

pH should cause a +59.16mV potential change. This relationship degrades over the lifetime of


the electrode and requires calibration. Additionally, pH electrodes are given to alkaline and acid

error where the pH versus potential relationship for the electrode is not linear at pH extremes.

Extreme pH error is not expected to affect this project, as it seeks to arrive at a neutral pH. The

sensitivity of the electrode used in this project was verified using a Fisher Scientific Accumet

Basic AB40 meter. The resulting relationship is shown in figure 8, where the slope is -57.97mV

per ph unit (at room temperature, ~23°C).

Figure 8: pH electrode sensitivity when connected to a standard high input impedance pH meter.

The voltage was obtained in the neutralizer system after connecting the pH electrode to

the NI ELVIS II+ and computer controller. Figure 9 gives the average ± standard deviation

calibration of the electrode corresponding to the pH of the buffer solutions in which the electrode

was placed. The potential change, from the slope of the calibration curve, and its relationship to

pH change was opposite of what it theoretical behavior. Additionally the voltage signal obtained

was not stable, as it had a large amount of noise. Still, the voltage signal changed depending on

the change in the solution’s pH. With an assumed full-scale analog voltage range output of

±11V and an ADC the resolution of 16 bits, the resolution of the ADC was determined to be

0.336mV. These variances represented a calibration and/or a data-acquisition error. Based on

this signal, and calibration, the feedback control of pH was still able to be achieved by using the

voltage calculated at 6.5 pH and setting the pumps to turn on or off based on the voltage being

higher or lower than this value.

Figure 9: pH electrode sensitivity when connected to the project’s DAQ controller.

y = -0.058x + 0.3998

R² = 0.9933

-0.4

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0

0.2

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0.6

0.8

1

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Po

tenti

al (

V)

pH

y = 0.1772x - 0.9207

R² = 0.9978

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Figure 10 depicts the voltage signal obtained by the neutralizer device as well as the

resulting signal change when the electrode sensor was alternated between a neutral and acidic

buffer. As the sensor was alternated around neutral and acidic buffers, the water and base pumps

switched on to off, respectively, as the signal passed above or below 0.23V, correlating to 6.5

pH.

Figure 10: Representative voltage vs. time data acquired after alternating the pH electrode between pH

7.00 and 4.01 buffer solutions. The sine wave data is given in light grey with the black line indicating the

moving average (period = 10). The left vertical red line indicates the time at which the electrode was

moved from the pH 7.00 buffer to the pH 4.01 buffer. The right vertical red line indicates the time at

which the electrode was moved from the pH 4.01 buffer, back to the pH 7.00 buffer.

Similarly, when the neutralization chamber was completely setup, an acidic chitosan

sponge was placed into the neutralization chamber and the resulting voltage change was recorded

and is shown in figure 11. The chitosan sponge was not able to be completely neutralized using

this method. Using this automatic neutralization method results in a brief drop in pH (depending

on the sponge size to be neutralized). After this, the pH remains stable at above the chosen 6.5

pH set point. The system was confirmed to be functioning due to the direct disturbance of the

neutralization container’s solution by physically adding a concentrated acidic solution. Due to

the high porosity and interconnectivity of the chitosan sponge (figure 12), there is assumed to be

inadequate fluid flow through the sponge and therefore internal sections of the chitosan sponge

were not neutralized.

-0.30

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Figure 11: Voltage vs. time data acquired from the pH electrode after placing an acidic chitosan sponge

into the neutralization chamber. The sine wave data is given in light grey with the black line indicating

the moving average (period = 10). The chitosan sponge was placed in the chamber at 0 seconds. The

vertical blue lines indicate the length of time that the basic solution pump was active (~3.5 seconds).

Figure 12: Scanning electron microscopy image of a chitosan sponge.

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Time (seconds)


In order to improve upon this system, a simple mechanical compression device could be

added to the neutralization container design, which could also be automated to compress

periodically throughout the neutralization (as seen in figure 13). The addition of mechanical

compression should provide sufficient fluid flow and exchange to neutralize the sponge. Other

design improvements could also be made, such as minimizing the neutralization container

solution volume, minimizing the connection lengths between the reservoirs and the neutralization

container, and the pH electrode could be placed in the solution flow escape line, all in order to

improved pH detection and decrease system response time. Implementing these design changes

could induce large step changes in the measured pH immediately after compression. It may then

be beneficial to add a PID controller to the feedback system to reduce the steady state error

between the desired pH and the actually pH as well as further improve the system response time.

Figure 13: Improved Design Layout

Errors in this system are primarily measurement errors, specifically signal acquisition

from the pH sensor. Signal output to the pumps was designed to be either on or off at a set motor

speed determined by the voltage output. Neither the exact speed of the motor or the response

time from signal acquisition to pump activation is critical towards the overall purpose of the

neutralizer. However the ability to detect the change in pH as related to the obtained voltage

signal was significant towards providing a neutralized chitosan sponge.

Additional improvements could be made to the detector in the system. As it stands, the

obtained sensor relationship between the pH and the voltage signal produced cannot be

definitively explained. Many possible sources for the changes can be identified, such as the wire

adaptor connections made to connect the sensor to the DAQ analog input, or an error in the

software signal analysis where signal conditioning could have been applied using the LabVIEW

program to correct for the signal relationship. A solution to this problem would be to us DAQ

device with an even higher input impedance. Another solution to this problem may be to utilize

the standard, high input impedance, pH meter to process the analog voltage signal into a digital

pH signal which could be directly connected as a digital input to the neutralizer’s control

software program.

However, an additional problem with the signal acquisition was the large amount of

signal noise. This problem could be easily remedied by using the pH meter as an analog to digital

converter, due to it having minimal issues with the sensor’s data processing (figure 8). If the

current automatic sponge neutralizer setup was kept, there are several steps that could be taken to

reduce noise in the input signal. The connecting wires could be kept as short as possible and

away from interfering noise sources. The wire connections could be shielded and the wire pairs


kept twisted as much as possible along their lengths. If the noise continued and could not be

remedied by using the digital input signal, then filtering or smoothing the analog signal should be

considered. There are other errors associated with the neutralizer system, such as those involved

with all electrical systems where there are unaccounted for, or unknown resistances and

interferences in the wires and in the resistors.

The long term use of the designed system would require recalibration of the pH electrode

in order to adjust for drift in the electrode. However, it was shown that the pH of the system

could be controlled using the proposed method for a short duration without consideration of

electrode drift. The system is expected to function over the full range of pH, especially since

detecting the full pH is, at this point, unnecessary. All that is truly necessary in this system is to

detect whether the sensor’s voltage signal is above or below the set point corresponding to a pH

of 6.5. Depending on the value of the signal obtained the pumps are either switched on or off.

Detecting the full pH range could be important if the pumping speeds were set to correspond to

the pH values, however this would require programming outside the scope of this project. When

used to neutralize a chitosan sponge, the system usually remained above the 6.5 pH set point and

was not completely effective at neutralizing the sponge interior, with the surrounding solution

remaining neutral. The cost of building such a device without any of the necessary materials

ranges in the thousands of dollars depending on the quality of products purchased.

This system is limited by the approach taken to achieve the signal. If altered to a design

that provides a signal with less noise and interference along with one that establishes the

appropriate relationship between the pH and voltage detected, the ability to provide a neutralized

sponge would be greatly improved. As it currently operates, due to signal noise, the pumps

cannot detect the neutral pH with enough resolution. As a result, it is expected that a signal as

low as 5.6 pH (based on the calibration and signal amplitude) would be considered neutral by the

system, when it remains acidic. This system was also limited by a design flaw which does not

include the ability of the neutralization fluids to flow directly through the highly porous chitosan

sponge device. A design change to force fluid flow through the sponge would also give the

ability to provide a more assuredly neutralized sponge. As a result of this limitation, the current

system is unable to neutralize the internal portions of a chitosan sponge, and reports a neutral

solution as a result.

The purpose of this system is to have the resulting pH of the neutralizing solution to be

greater than or equal to the set input pH, and consequently resulting in a neutralized sponge. The

pH electrode converts the number of hydronium ions in the solution to a voltage. The

neutralization solution pumps switch on or off determined by the input pH electrode voltage in a

feedback path. The system operates to drive the error between the desired set pH and the actual

pH to zero. When the input and output match, the error will be zero, and the neutral pH solution

pump will remain switched on until stopped by an operator. Thus, when the currently designed

system is running, one pump always remains on. As long as the voltage (and pH) detected from

the electrode is below the set value, the basic solution pump will turn on, and whenever the

voltage is greater than or equal to the desired set voltage, the neutral solution pump will turn on.

Conclusion

This project demonstrated a neutralization process alternative to the manual procedure

currently used in manufacturing of chitosan sponges for research purposes. The fundamental

concept of controlling pH of a solution in order to neutralize a hydrated, acidic sponge was


successful, although some design and instrumentation changes are needed if this project is to be

truly successful. Once these limitations are addressed, then the development of this system could

be justified for the large scale manufacturing of more consistent and controlled, neutralized

chitosan sponges.

References

1. Abdou ES, Nagy KS, Elsabee MZ. Extraction and characterization of chitin and chitosan

from local sources. Bioresour Technol. Mar 2008;99(5):1359-1367.

2. Nunthanid J, Laungtana-Anan M, Sriamornsak P, et al. Characterization of chitosan

acetate as a binder for sustained release tablets. J Control Release. Sep 14 2004;99(1):15-

26.

3. Khor E, Lim LY. Implantable applications of chitin and chitosan. Biomaterials. Jun

2003;24(13):2339-2349.

4. Puttipipatkhachorn S, Nunthanid J, Yamamoto K, Peck GE. Drug physical state and

drug-polymer interaction on drug release from chitosan matrix films. J Control Release.

Jul 10 2001;75(1-2):143-153.

5. Bravo-Osuna I, Vauthier C, Farabollini A, Palmieri GF, Ponchel G. Mucoadhesion

mechanism of chitosan and thiolated chitosan-poly(isobutyl cyanoacrylate) core-shell

nanoparticles. Biomaterials. Apr 2007;28(13):2233-2243.

6. Cardenas G, Anaya P, von Plessing C, Rojas C, Sepulveda J. Chitosan composite films.

Biomedical applications. J Mater Sci Mater Med. Jun 2008;19(6):2397-2405.

jks2012june - level a project report (3)

Documents

automatic neutralizer

background chitosan

use of chitosan

list of figures figure

project report page

chitosan sponges

chitosan sponge sem

neutralizer prototype