UPTEC F10 028

Examensarbete 30 hpMaj 2010

Spectrophotometric measurement automatization for the analysis of enzymatic processes

Karolina Elisabeth Nilsson

Teknisk- naturvetenskaplig fakultet UTH-enheten Besöksadress: Ångströmlaboratoriet Lägerhyddsvägen 1 Hus 4, Plan 0 Postadress: Box 536 751 21 Uppsala Telefon: 018 – 471 30 03 Telefax: 018 – 471 30 00 Hemsida: http://www.teknat.uu.se/student

Abstract

Spectrophotometric measurement automatization forthe analysis of enzymatic processes

Karolina Nilsson

This thesis work consisted of the development of a virtual instrument that automatesenzyme activity measurements and spectrum measurements with thespectrophotometer UVmini-1240. The purpose was to expand the functionality of theinstrument, to eliminate the human error and to decrease the amount of time spenton measurements. A PC was connected to the UVmini-1240 via a RS-232C interface and the cellposition and temperature was regulated with a CPS-240A controller. The newinterface allows all the parameters to be set in the same place. It allows a visualizationof the continuous monitoring of the sample absorbance and the option to save thedata for post-processing. Also a module for measuring the spectrum of a sample inthe wavelength range of 190 nm to 1100 nm is included. The graphical programminglanguage LabView was used to develop the virtual instrument. This thesis work also contained measurement series of the catalase enzyme activity.These were carried out to determine the best storage temperature for the catalasesolution and to determine the optimal surrounding temperature for the highestactivity in the catalase solution. The conclusions were that the activity does notchange considerably the first week of storage, not matter the temperature, and thatthe activity goes down when the surrounding temperature reaches above 30° C.These measurements were part of a bigger project to develop an ultrasonic methodfor measuring enzyme activity at the Institute of Acoustics at C.S.I.C in Madrid.

Sponsor: Instituto de Acústica, C.S.I.CISSN: 1401-5757, UPTEC F08 000Examinator: Tomas NybergÄmnesgranskare: Tadeusz StepinskiHandledare: Luis Elvira

Contents 1 Introduction …………………………………………………………………………………………….......... 1

2 Methodology and Theory ………………………………………………………………………........... 4

2.1 Instrumentation Control ………………………………………………………………………........ 4

2.1.1 Spectrophotometry ………………………………………………………………………........... 4

2.1.1.1 Measurement principle …………………………………………………………………… 4

2.1.1.2 Single beam spectrophotometer …………………………………………………… 4

2.1.1.3 UVmini-1240 instrument description …………………………………………….. 5

2.1.1.4 Instrument functionality ……………………………………………………………….. 6

2.1.2 Programming ……………………………………………………………………….................... 7

2.1.2.1 Graphical programming ……………………………………………………………….. 7

2.1.2.2 LabView ………………………………………………………………………................... 8

2.2 Enzymatic Characterization Methodology ………………………………………………….. 10

2.2.1 Enzymes ………………………………………………………………………............................ 10

2.2.2 Catalase ………………………………………………………………………............................ 10

2.2.2.1 Applications ………………………………………………………………………............... 11

2.2.3 Enzymatic Activity Measurements …………………………………………………………. 11

2.2.3.1 Enzyme activity ………………………………………………………………………........ 11

2.2.3.2 Catalase activity test preparations ………………………………………………… 12

2.2.3.4 Spectrophotometer measurement procedure ……………………………… 14

3 The Virtual Spectrophotometer Interface ……………………………………………………….. 16

3.1 Communication with the UVmini-1240 ……………………………………………………… 16

3.1.1 Connecting to PC ………………………………………………………………………............. 16

3.1.2 Communication protocol ……………………………………………………………………… 17

3.1.3 Write command; Protocol A …………………………………………………………………. 17

3.1.4 Read command; Protocol B and Protocol B’ …………………………………………. 18

3.2 The Photometric Mode ………………………………………………………………………........ 18

3.2.1 The GUI ………………………………………………………………………............................ 19

3.2.2 The flow chart of the photometric mode …..…………………………................. 21

3.2.3 The spreadsheet file ………………………………………………………………………........ 23

3.3 The Spectrum Mode ………………………………………………………………………............ 24

3.3.1 The GUI ………………………………………………………………………........................... 25

3.3.2 The flow chart of the spectrum mode ..…………………………………................. 26

3.3.3 The spreadsheet file ………………………………………………………………………....... 28

3.4 The SubVIs ………………………………………………………………………........................... 28

3.4.1 writeVI ………………………………………………………………………............................ 29

3.4.2 readVI ………………………………………………………………………............................. 29

3.4.3 Other subVIs ………………………………………………………………………................... 30

4 Experiments and Results ………………………………………………………………………........... 32

4.1 Data Analysis for Catalase Activity …………………………………………………………….. 32

4.2 Measurements in Photometric Mode ……………………………………………………….. 35

4.2.1 Change in catalase acitivty due to storage temperature ………………………. 35

4.2.2 Changes in catalase activity due to surrounding temperature ……………… 40

4.3 Measurements in Spectrum Mode ……………………………………………………………. 42

4.3.1 Spectrum measurements of hydrogen peroxide ………………………………….. 43

4.3.2 Spectrum measurements of different sugars ……………………………………….. 44

4.4 Discussion …………………………………………………………………………………………………. 46

4.4.1 The storage temperature measurements …………………………………………….. 46

4.4.2 The surrounding temperature measurements …………………………………….. 46

4.4.3 Accuracy of the curve fitting ………………………………………………………………… 47

5 Conclusions ………………………………………………………………………................................... 48

5.1 The Program ………………………………………………………………………........................ 48

5.1.1 The photometric mode ………………………………………………………………………... 48

5.1.2 The spectrum mode ………………………………………………………………………........ 49

5.2 The Contributions ………………………………………………………………………................ 49

5.3 Future Work ………………………………………………………………………......................... 49

References ………………………………………………………………………......................................... 51

Appendix A …………………………………………………………………………………………………………….. 52

1

Chapter 1

Introduction

When purchasing traditional hardware instrumentation it will most likely be hard-coded to

work in a certain way. The measurement procedure is pre-defined. Virtual instrumentation is

the name for customizing software for measurement instruments to better suit the needs of

the user. The software interface between the measurement instrument and the PC is called

a Virtual Instrument. A Virtual Instrument is created to extend the functionality of the

measurement equipment. By the means of programming the possibilities of a given

instrument are expanded, allowing measurements that could not be obtained before. To aid

in creating these Virtual Instruments are a number of graphical programming packages, for

example National Instrument’s LabVIEW, which has been used in this project.

Spectrophotometry is the analysis technique using the electromagnetic spectra. There are

instruments called spectrophotometers that use this technique for measuring and analyzing

transparent, liquid samples. A spectrophotometer sends light of a certain wavelength

through a sample and measures the absorbance or transmittance of the sample. This can

then give information of the sample’s components and their amounts, since different

molecules absorb light at different wavelengths. The spectrophotometer is commonly used

to measure enzyme activity.

Enzymes are proteins that catalyze a variety of chemical reactions, [1]. In the cells of an

organism a lot of reactions take place and to occur in a significant rate almost all processes

in biological cells need enzymes. A reaction caused by an enzyme can be millions of times

faster compared to an un-catalyzed reaction. The enzyme itself does not get damaged or

distorted by the process, which means it can keep on reacting with another substrate after

finishing with one. It is possible to affect the activity of the enzymes with other molecules

(inhibitors or activators) or by outer circumstances such as temperature, pH or

concentration of the substrate.

The enzyme of interest for this project is called catalase. Catalase separates hydrogen

peroxide into water and oxygen gas. It has many applications and can, for example, be used

in the textile industry to remove hazardous hydrogen peroxide from the fabrics after

bleaching, [5]. Since catalase can be very useful both for commercial and industrial purposes,

[2], it is natural that there is an interest to characterize the activity of this enzyme.

The most common way to determine the activity of catalase is with a spectrophotometer.

Hydrogen peroxide absorbs light at the wavelength of 240 nm but water and oxygen gas do

not. This can be seen clearly in figure 29 and 30, which shows a spectrum of the hydrogen

peroxide solution. For this reason it is possible to observe the activity of catalase by looking

2

at how the hydrogen peroxide levels are declining, [12]. A bit of theory and methodology

behind the spectrophotometer and the measurements can be seen in chapter 2.

New research has shown that it is possible to use ultrasonics to analyze liquids, [13]. This can

be used to measure the activity of catalase. By transmitting an ultrasonic wave through the

liquid, and measuring the backscatter of the oxygen bubbles, it is possible to get information

about the activity. In this case the quantity of the bubbles, created by the oxygen gas that is

being produced by the decomposition of the hydrogen peroxide, is being measured. The

solution is being jellified for the bubbles to stay inside the medium.

The Ultrasonic Department of Instituto de Acústica of C.S.I.C1 in Madrid wants to find an

ultrasonic method to analyze catalase solution that will exceed the sensitivity of the

spectrophotometer. Tests with identical samples will be carried out both with the

spectrophotometer and the ultrasonic measurement equipment to compare the results of

the new technique with the already acknowledged one.

In order to start that project, information about the catalase behavior needs to be collected.

But the spectrophotometer only shows the value of the absorbance in real-time on its

monitor. The measuring procedure and enzyme activity analysis has up till now been very

basic; the only data collected, by hand, is the time between the absorbance values 0.45 and

0.4. If a graph over the activity is wanted someone has to write down all the values, with a

certain time interval, and then enter them manually into the computer and make a graph

out of it. This is very time consuming and might not be very correct due to the factor of

human error. Also it is impossible to look at the velocity in the beginning of the reaction,

which is informative when looking at enzymatic reactions.

A Virtual Instrument is needed for the spectrophotometer, to automate the measurement

procedure. This will improve the instruments performance by adding data storing

possibilities for data post-processing, data acquisition over long time periods and visual

measurement results. Enzymatic reactions can be very fast and to analyze them rapid data

acquisition is needed. With a Virtual Instrument the instruments expanded functionality will

be better suited for the rapid data acquisition needed for measuring enzyme activity. Also

the use of the spectrophotometer’s monitor and small keyboard will be substituted by a

user-friendly GUI, gathering all the spectrophotometer’s parameters. This application will

not only be useful for catalase measurements but for many other enzyme measurements as

well.

The first part of the thesis work was to develop the software to communicate with and

control the spectrophotometer. Initially there was no software and no PC connected to the

spectrophotometer. The programming of the software was done in LabVIEW. All the

relevant parameters of the spectrophotometer can be set with the controller software and

also the parameters for the functions extended by the Virtual Instrument, i.e. parameters for

the repeated data acquisition such as time between measurements and experiment length. 1 Consejo Superior de Investigaciones Científicas. A governmental agency that gathers the scientific institutes in

Spain.

3

The measurement results are shown both in a graph and a data table and all the

measurement data is saved on the hard drive of the controller PC for later considerations.

The program consists of two parts: the photometric mode and the spectrum mode. The

photometric mode measures the absorbance at a certain wavelength and produces a graph

that shows the declination of the hydrogen peroxide, see section 3.2. The spectrum mode

measures the absorbance of a range of wavelengths, see section 3.3. The graph produced by

the measurement data shows the spectrum of the sample.

The second part was to do catalase activity measurements to evaluate the possibilities of

storing the catalase solution for a longer period of time without losing too much of the

activity. This is important to know when doing a series of measurements that last for more

than one day. Also a series of measurements were conducted to see what the optimal

measurement temperature is for the bovine liver catalase. Measurements to calculate the

amount of enzyme units in different catalase solutions have been carried out for the purpose

of the ultrasonic measurements. These measurements were done in the photometric mode,

see section 4.2.

Spectrum measurements of the hydrogen peroxide solution before, during and after

hydrolysis were conducted. Also some measurement for other projects such as the spectrum

of different sugars and amino acids were carried out. These measurements were done in the

spectrum mode, see section 4.3.

After my work has been done the ultrasonic measurements started. My program and

measurements assist this project with information about catalase such as optimal storing

temperature, measurement temperature and measurements for calculating the units of

enzymes per ml in various catalase solutions.

4

Chapter 2

Methodology and Theory

2.1 Instrumentation Control

This section explains how the instrument that is being used works as well as the means of

controlling it.

2.1.1 Spectrophotometry

In the project the method of spectrophotometry is used to measure the catalase enzyme

activity. A spectrophotometer from Shimadzu called the UVmini-1240 has been used, see

figure 2. The controller CPS-240A is connected to the UVmini-1240 to be able to control the

sample holder and the temperature of the samples. The software will be designed to control

the UVmini-1240 and the CPS controller to take measurements automatically. In this section

some background to this type of measurements is given.

2.1.1.1 Measurement principle

Spectrophotometry is the measureable analysis technique using the electromagnetic

spectra. It deals with the ranges of wavelengths such as near ultraviolet, near infrared and

visible light. A device called a spectrophotometer is used to measure the absorbance or

transmittance through a liquid sample. The spectrophotometer takes a measurement at a

specific wavelength and it is possible to choose any wavelength in the spectrophotometers

register.

There are two popular ways to do this; with single beam and with double beam. The double

beam sends a beam through a blank reference sample and one through the sample to be

measured. The single beam measures the difference between the light intensity before and

after passing through the sample. The UVmini-1240 is a single beam spectrophotometer.

2.1.1.2 Single beam spectrophotometer

In the single beam spectrophotometer there is one or more light sources that produce a

range of wavelengths. In figure 1 the general structure of a single beam spectrophotometer

is shown.

5

Depending on the desired wavelength a suitable lamp is chosen. The light hits the

monochromator and gets diffracted into a spectrum of wavelengths. There is an adjustable

aperture that only lets the desired wavelength through the glitch so that it can pass through

the sample. The light sensor then measures the intensity of the light and the signal is being

amplified and displayed on the monitor. Usually an auto zero is made with a blank sample,

before measurements, to get the sample intensity to be measured in reference to the blank.

That makes it possible to measure only the absorbance of the hydrogen peroxide, by first

doing an auto zero on a pure sample of the buffer that it is diluted in.

2.1.1.3 UVmini-1240 instrument description

The UVmini-1240 has the wavelength range of 190 nm – 1100 nm, with a resolution of 5 nm.

It is a single beam spectrophotometer; a description of this can be seen in section 2.1.1.2.

The light sources are a 20W halogen lamp and a deuterium lamp. The detector is a silicon

photodiode. The UVmini-1240 is connected to a CPS controller that can control the cell

position and the cell temperature, see figure 2. The CPS controller has 6 cells that can

contain a sample, and an engine to move the sample holder to the desired positions. It has a

thermoelectrical temperature controller that is attached on the sample holder for

temperature regulation of the samples. The little square-shaped glass container that the

sample liquid is placed in is called a cuvette.

It is possible to connect the UVmini-1240 to a PC and control it with the pre-coded

commands. The communication is made through a RS-232C interface. For a full list of the

hardware and software specifications see Appendix A.

I0 A 0.550

Light source

Adjustable aperture Light sensor Output

Amplifier

Cuvette

Sample

Monochromator

I

Figure 1 The struture of the single beam spectrophotometer.

6

Figure 2 Right: the UVmini-1240. The measurements can be seen on the monitor and it is controlled by the buttons just below the monitor. Left: the CPS controller that controls the cell position and the temperature of the cell holder.

2.1.1.4 Instrument functionality

The UVmini-1240 has a few different modes that can be shown on the monitor of the

spectrophotometer. It can be controlled directly with the buttons of the keyboard under the

monitor. The different modes are:

Photometric mode

Spectrum mode

Quantitation mode

Utilities mode

PC control mode

The modes that are interesting for this project are the photometric mode, spectrum mode

and PC control mode, but a brief presentation of all the modes will be given to give an idea

of what the UVmini-1240 does. The main operative modes are the photometric mode and

the spectrum mode, which carry out the measurements. The other modes are used for post-

processing and other secondary functions.

Photometric mode

This is the fixed wavelength measurement mode. It is possible to measure the absorbance or

the transmittance at a fixed wavelength. By repeating a measurement it is also possible to

create a table or graph of the measurement result2.

Spectrum mode 2 By hand. There will not be a graph or table displayed on the monitor.

7

In this mode the spectral measurements are performed. It is possible to choose between

measuring absorbance, transmittance or single beam energy. The measurement is being

done in the chosen wavelength interval. In the spectrophotometer it is possible to use

various data processing such as scale change and peak/valley detection.

Quantitation mode

This is the mode in which unknown samples are quantitated by creating a calibration curve

from standard samples. There are 3 types of quantitation; one-, two- and three wavelength

method. There are three methods for creating calibration curves; K-factor method, single

point calibration curve method and multi-point calibration curve method.

Utilities mode

This is the mode for setting the instrument’s operating parameters, such as the light source

switching and printer selection. The parameters that can be set here are also shared with

other modes. They are being stored in memory even when the spectrophotometer is turned

off.

PC control mode

This mode is used when the UVmini-1240 is controlled by an external computer. There is a

set of commands that can be used in the communication. An RS-232C interface is used to

communicate.

2.1.2 Programming

For the project software will be developed to communicate with and control the UVmini-

1240. The program should control the sample holder’s position, be able to set the

parameters of the UV-1240 and retrieve the data. The language that is most adapted to this

kind of communication is LabView, which is a graphical programming language.

2.1.2.1 Graphical programming

The definition of a graphical programming language is that the programmer can construct

the program by manipulating graphical components instead of specifying everything

textually. They are mostly based on the idea of boxes and wires/arrows. The boxes are like

8

the functions in a traditional programming language and they are connected with the wires

and arrows, which represent the relationship between the functions. Through the wires the

boxes are given input parameters and the outputs are taken. There are many graphical

programming languages out there, and they are specialized in different areas.

2.1.2.2 LabView

LabView has been developed by the company National Instruments. It is mainly designed for

engineers and scientists. It is most commonly used for data acquisition, instrument control

and industrial automation. LabView is based on boxes that are connected with wires. The

boxes are like functions, a piece of code that has an input and an output.

LabView comes with an extensive set of functions and graphical components. It is based on

the idea of having a front panel and a block diagram. The front panel is the graphical

interface, which in LabView is very easy to create, the components are just placed where

they are wanted. The components in the front panel that represent or visualize a value are

also shown in the block diagram. It is in the block diagram all the coding is done and where

manipulation of the front panel components is done. In figure 3 and 4 a front panel and its

block diagram is shown. A program made with LabView is called a Virtual Instrument or a VI.

LabView has some standard programming functions, to be able to create dynamic programs.

The three blocks are: while-loop, for-loop and case-structure. They are graphically

represented by a frame where the code to be run is put inside. Case-structure is like an if-

structure but more than 2 alternatives are possible. With a feature called shift-register it is

possible to transfer parameters between the loops, see figure 5.

Figure 3 The front panel of a small LabView program.

Figure 4 The block diagram of the small program in figure 3. Numeric is a controller and Slide is an indicator. They are both front panel components.

9

When making a big program it is necessary to do everything that is possible to simplify the

code and make it more understandable. As in all programming languages it is smart to make

functions out of code that is used many times and can communicate with a few input and

output parameters. This way the code will be easier to grasp. In LabView these functions are

called subVIs. They are Virtual Instruments within the main Virtual Instrument. In figure 6 it

can be seen how this would look in the block diagram of figure 4. The middle part of the

block diagram has been turned into a subVI.

Figure 6 The middle part of the block diagram in figure 4 has been turned into a subVI that has a numeric as input and a numeric as output.

Figure 5 The arrows on the sides are called shift-registers. They make it possible to transfer the data between the loops.

10

2.2 Enzymatic Characterization Methodology

This section explains the use of enzymes and explains the measurement methodology and

the reasons for measuring the enzyme activity.

2.2.1 Enzymes

Enzymes are proteins that speed up chemical reactions, [1]. They are made out of long linear

chains of amino acids that fold up to produce a 3-dimensional structure. It is this structure

that decides the function of the enzyme, although predicting the function of an enzyme just

by knowing the structure is a problem yet to be solved.

The molecules in the beginning of a process in the enzymatic reaction are called substrates,

and the enzyme parts them into different molecules called products. Enzymes are not

changed by the reactions they catalyze so they can keep on reacting with a new substrate

after finishing with one.

The enzyme is usually distinctly larger than the substrate and only a small part is directly

occupied by the catalysis. It is possible to denature3 enzymes, i.e. to unfold and inactivate

the proteins. It can be done by heating or adding chemical denaturants. For some enzymes

this process can be reversible.

Enzymes are used for commercial purposes on a large-scale, [2] [3]. They can be used, for

example, to break down the protein in meat to make it tender, make juice clearer with the

enzyme pectinase and resolve stains in fabric by using enzymes to break down the fat and

proteins. Even the very old method of fermentation to produce alcohol is due to the enzyme

in the yeast breaking down the sugar into ethanol [4].

2.2.2 Catalase

Catalase is an enzyme that functions to decompose hydrogen peroxide to oxygen gas and

water molecules. It is common to find in most living organisms that are exposed to oxygen.

When cells perform metabolic processes hydrogen peroxide can be a harmful by-product.

The cells then use catalase to speed up the process of breaking down the harmful hydrogen

peroxide to the less dangerous oxygen gas and water molecules. The catalase reaction of the

decomposition is:

2𝐻2𝑂2 → 2𝐻2𝑂 + 𝑂2

3When enzymes denature they lose their active state.

11

The catalase used in this project is extracted from bovine liver. It should be stored in the

freezer to maintain activity. It is soluble in 50 mM potassium phosphate buffer with a pH of

7.0, [5]. The EC number4 of this enzyme is 1.11.1.6.

2.2.2.1 Applications

There is a need for catalase in many industries. This is mainly because catalase decomposes

hydrogen peroxide which is a harmful molecule for living organisms. It is necessary to

remove it from products after use. In high concentrations it is corrosive and dangerous when

getting in contact with the skin or eyes.

It has a large range of applications. For example in the textile industry it is used to remove

the dangerous hydrogen peroxide from fabrics after they have been bleached [5]. In the

food industry it is used to remove the hydrogen peroxide from the milk before making

cheese, [6]. Also it is used in wrapping paper for food products to prevent them from

oxidizing.

2.2.3 Enzymatic Activity Measurements

It takes a very small amount of enzymes to prepare an enzyme solution with the wanted

activity. Also the proteins of the enzyme can be damaged or unfolded in the process of

producing a solution or during the storage of the enzyme. It is very hard to weight these

small amounts that are needed and the activity of the enzyme is not completely known. This

means that after making a solution the activity will be a bit uncertain. Other methods of

controlling the activity of the enzyme solution are needed. In this project spectrophotometry

is used, but there are a lot more methods out there.

2.2.3.1 Enzyme activity

The amount of enzymes can be expressed either in molar amounts, as all chemicals, or in

terms of activity, as enzyme units. The definition of enzyme activity is the number of moles

of substrate converted per unit time. The unit to measure catalytic activity is called katal.

The definition of katal is; the catalytic activity that will raise the rate of reaction by one mole

per second in a specified assay5 system, [7]. But this unit is impractically large so the most

commonly-used value to measure enzyme activity is 1 enzyme unit (U) = µmol min-1 which is

16.67 nkat.

4 The Enzyme Comission Number is the numerical classification scheme based on the reactions the enzyme

catalyzes. 5 An enzyme assay is a laboratory method for measuring enzymatic activity.

12

There are many types of enzyme assays. They can be divided into four groups of

experiments; initial rate experiments, progress curve experiments, transient kinetics

experiments and relaxation experiments. The different methods for activity measurements

are also divided into two groups; continuous assays and discontinuous assays. Continuous

assays give a continuous reading of the activity. With discontinuous assays samples are

taken, the reaction is stopped and the concentration of the substrate and product is

measured.

Continuous assays

There are many different continuous assays. Spectrophotometry is for example a continuous

measurement method. A few other methods are fluorometric, calorimetric and

chemiluminescent. A fluorometric assay uses the fact that some molecules emit light of one

wavelength after absorbing light from another. Fluorometric assays look at the difference

between the light emitted from the substrate and the light emitted from the products, to

measure enzyme activity [8]. Calorimetric assays measures the heat released or absorbed by

chemical reactions. This is the most general assay, since many reactions involve a change in

heat, and can be used when no other assay is applicable [9]. Some chemical reactions

produce light that can be measured. This can be measured with a chemiluminescent assay

and can give very precise results [10].

Discontinuous assays

The discontinuous assays are radiometric and chromatographic. There are many ways of

using radiometry for enzyme assays but the principle is to monitor the conversion of

radiolabeled substrate to labeled products [8]. Chromatographic assays measure the product

formation by separating the reaction mixture with chromatography. This is usually done with

HPLC (high performance liquid chromatography) [11].

2.2.3.2 Catalase activity test preparations

It is possible to use a spectrophotometer to measure the activity of catalase. What is being

measured is how rapidly the substrate gets parted into products. For catalase the substrate

is hydrogen peroxide. Hydrogen peroxide is absorbing light approximately at the

wavelengths between 200 and 250 nm, see figure 30 in section 4.3.1. The measurements are

carried out at wavelength 240 nm because at this specific wavelength neither the oxygen,

water nor catalase is absorbing light, [12].

13

To perform an activity measurement a solution with hydrogen peroxide is prepared and

placed in a cuvette in the sample holder of the spectrophotometer. Then the catalase

solution is added and the absorbance is monitored. Below is a detailed description of how to

prepare the reagents and to perform the measurement.

Potassium phosphate buffer

To do the measurements a 50 mM potassium phosphate buffer that has pH 7.0 at 25°C has

to be prepared. The bovine liver catalase is soluble in this buffer.

Start by taking 200 ml of deionized water. Add 1.361 grams of potassium phosphate. Stir the

compounds in the magnetic stirrer till the potassium phosphate is totally resolved. Then

adjust the pH by using the pH-meter and by adding KOH (potassium hydroxide) till the pH

reaches 7.0. This buffer is then used to produce the other reagents and also needed for

doing the auto zero in the spectrophotometer before starting measurements. The buffer can

be stored in the fridge.

Hydrogen peroxide solution

The hydrogen peroxide solution consists of concentrated hydrogen peroxide resolved in the

potassium phosphate buffer. The hydrogen peroxide solution should be 0.036% (w/w)6 of

hydrogen peroxide. The hydrogen peroxide used is 50% (w/w). So for 50 grams of potassium

phosphate buffer 0.036 grams7 of 50% (w/w) hydrogen peroxide is needed. This solution is

done by putting a beaker on a sensitive digital scale, zeroing the instrument, adding 50

grams of buffer, zeroing the instrument again and adding 0.036 grams of hydrogen peroxide.

It is then mixed thoroughly.

It is important that the solution is in the right absorbance interval. It should be between 0.52

and 0.55 absorbance units. To make sure that absorbance of the hydrogen peroxide solution

is in the wanted interval the solution is taken to the spectrophotometer to be measured. If

the absorbance is lower than 0.52 more hydrogen peroxide is added. If it is higher than 0.55

more potassium phosphate buffer is added. The hydrogen peroxide solution is always made

right before measurements.

6 percentage of the weight

7 (50 grams buffer)*(0.036%)*2 = 0.036 grams

14

Catalase solution

To prepare the catalase solution with an exact concentration is nearly impossible. A very

sensitive digital scale is necessary. The wanted solution contains 50 – 100 enzyme units per

ml, which is approximately 0.00025 grams of bovine liver catalase for 10 grams of buffer.

First a beaker with 10 grams of cold potassium phosphate buffer is prepared. A piece of

paper is put on the scale and the instrument is zeroed. The bovine liver catalase is taken out

of the freezer and a small amount is put on a piece of paper. Slowly catalase is added to the

piece of paper on the scale, till 0.00025 grams is reached. The measurement uncertainty is

very big here because even the possibility of the paper or the catalase absorbing humidity

from the air will alter the result. The piece of paper with the weighed catalase is taken and

the catalase added to the cold buffer reagent. It is then mixed thoroughly.

The catalase solution was always made right before every new test. In this thesis project it is

analyzed if it is possible to store the solution in the fridge or freezer and how long it takes

before a significant difference in activity is shown. This may save important amounts of

product.

2.2.3.4 Spectrophotometric measurement procedure

Without a program the measurement procedure for determining the catalase activity would

be performed on the monitor of the UVmini-1240. First the wavelength is set to 240 nm and

then a cuvette is put into the first cell of the sample holder. 3 ml of the potassium phosphate

buffer is added using a pipette. The measurement beam is set to the first cell. An auto zero is

performed on the sample, to use the buffer as the reference. The cuvette is emptied and

cleaned and put back into the first cell of the sample holder.

2.9 ml of the hydrogen peroxide is added to the cuvette. It is important to make sure that

the absorbance value is between 0.52 and 0.55. This is done by looking at the real time

absorbance value on the monitor of the spectrophotometer. Then 0.1 ml of the catalase

solution is added and mixed with the hydrogen peroxide solution. The real time absorbance

value is observed and a timer is started when the absorbance value reaches 0.45 and

stopped when the value reaches 0.4. This value, in minutes, is then used in a formula to

calculate the units of enzymes per ml.

The final assay concentration of 3 ml reaction mix is 50 mM of potassium phosphate, 0.035%

(w/w) hydrogen peroxide and 5 – 10 units of catalase. The unit definition of catalase is that

one unit will decompose 1.0 µM of hydrogen peroxide in pH 7.0 at 25°C, while the hydrogen

peroxide solution falls from 10.3 mM to 9.2 mM.

15

To calculate the units of enzymes per ml the following formula is used:

𝑈𝑛𝑖𝑡𝑠 𝑜𝑓 𝑒𝑛𝑧𝑦𝑚𝑒𝑠/𝑚𝑙 =(3.45) ∗ (𝑑𝑓)

(min)∗ (0.1)

3.45 = Corresponds to the decomposition of 3.45 micromoles of hydrogen peroxide in a 3

ml reaction mixture producing an increase in the absorbance from 0.45 to 0.4

absorbance units.

df = Dilution factor.

min = Time in minutes required for the absorbance to go from 0.45 to 0.4 absorbance

units.

0.1 = Volume (in milliliter) of enzyme used.

16

Chapter 3

The Virtual Spectrophotometer Interface

3.1 Communication with the UVmini-1240

The first thing to do to automatize the spectrophotometer is to connect it to an external

computer that is programmable. It is also important to know the communication protocols

so that communication can be conducted with the spectrophotometer in a way that it can

understand the commands that are being sent.

3.1.1 Connecting to PC

To control the UVmini-1240 by an external computer (PC) an RS-232C interface is used. The

specification for wiring the RS-232C interface that connects the UVmini-1240 to the PC is

shown in figure 7.

The part number of the RS-232C cable that is being used is 200-86408. 3 wires are being

used, 2 for input/output and 1 for ground.

D-sub 9-pin male

connector on PC D-sub 9-pin male

connector on Spectrophotometer

1

2

3

4

5

6

7

8

9

RXD

TXD

DTR

GND

DSR

DTS

CTS

3

2

7

RXD

TXD

Signal GND

Figure 7 Specification for connecting the PC to the UVmini-1240 with an RS-232C interface.

17

The configuration of the RS-232C port is as follows:

Transmission rate: 9600 bps

Data bits: 7 bits

Stop bit: 1 bit

Parity bit: Odd

3.1.2 Communication protocol

To communicate with the UVmini-1240, through the RS-232C port, it has to be set to the PC

Control mode. This is done on the command screen of the UVmini. When communicating

between the PC and the spectrophotometer there is one “listener” and one “speaker”. There

is a set of hexadecimal control codes to make sure that the communication in conducted

properly. These control codes are sent between the commands and data in a certain way.

The different procedures of sending commands, data and control codes are called protocols.

The control codes are: ENQ ($05) – Enquiry EOT ($04) – End of Transmission ESC ($1B) – Escape ACK ($06) – Acknowledge NAK ($15) – Negative Acknowledge NUL ($00) – Null

3.1.3 Write command; Protocol A

The write command makes it possible to set the status of the spectrophotometer. For

example moving the sample holder, setting the wavelength of the light, setting the

measurement mode etc… In figure 8 the time chart for Protocol A is shown.

Time

Write

status

s

E

N

Q

A

C

K

NUL

A

C

K

E

O

T

A

C

K

UV

PC

COMMANDS

Figure 8 A time chart of Protocol A, the protocol for writing a command to the UVmini-1240. ENQ, ACK, NUL and EOT are the control codes for this procedure.

18

3.1.4 Read command; Protocol B and Protocol B’

The read command makes it possible to know the status of the spectrophotometer.

Information such as the current data value and cell position can be retrieved. The Protocol B’

is for obtaining the data saved in the data storage buffer of the spectrophotometer, when a

scan measurement or time scan measurement has been taken. Figure 9 shows the time

chart of Protocol B and B’.

3.2 The Photometric Mode

This mode is designed to control the spectrophotometers photometric measurements. The

spectrophotometer itself does not give a table of data or a graph of the measurements. It

merely shows the real time measurement value on its’ display monitor. It is a bit of a hustle

to set the parameters of the spectrophotometer since its monitor and keyboard are rather

primitive. The photometric mode in the program can set all the parameters that are

available in the spectrophotometer’s photometric mode. It can, for example, carry out an

auto zero on a blank sample, choose the type of measurement or set the wavelength in

which the measurements are being performed. The program contributes in this aspect by

giving a clearer overview of the parameter settings.

The types of measurements available in this mode are absorbance and transmittance. The

transmittance T is defined as T = I/I0, which is the fraction of the incident light passing

through a sample. I is the intensity that passes through the sample and I0 is the intensity of

the sent light. The absorbance A is defined as A = 𝑙𝑜𝑔(1

𝑇).

Figure 9 A time chart of Protocol B and B’, the protocol for reading data from the UVmini-1240. ENQ, ACK, NUL and EOT are the control codes for this procedure.

Time

There are also repeating commands in this

portion. This is Protocol B’.

Read

Status

E

N

Q

A

C

K

NUL

A

C

K

DATA

A

C

K

UV

P

C

COMMANDS

A

C

K

E

O

T

A

C

K

N

U

L

E

N

Q

19

Besides gathering all the available parameters for easy setting with a user friendly interface

the program also contributes to new functions of the spectrophotometer. It is possible to

start a measurement series with the program where the program retrieves data from the

spectrophotometer at a certain time interval. The time interval and the length of the

experiment are parameters in the program. The program shows a real time graph of the data

measurements vs. time and also a table with the numerical data. This feature extends the

function of the spectrophotometer extensively. Instead of having a person monitoring the

screen and writing down the values with a certain time interval, the program produces a real

time graph and instantly gives the tester an easy visual overview of the data. This both saves

time and eliminates the risk of human error. Before starting a measurement a file dialogue

pops up and a target file for the data can be chosen. All the data taken during the

experiment will be saved in this spreadsheet file, making later consideration and post-

processing of the data possible. The data is stored continuously onto the file along the

experiment, which means that the experiment can be interrupted at any time without losing

information already retrieved.

To maximize the efficiency all of the 6 cells in the sample holder can be used for synchronous

measurements. That means that the program gives the option to measure 1-6 samples in the

same measurement series. Any combination of cells can be used. The program changes the

position of the sample holder by sending commands to the CPS controller. This automation

is another example of the timesaving qualities of the program.

3.2.1 The GUI

The graphical user interface of the photometric mode can be seen in figure 10. Here comes

an explanation for all the parameters that can be defined:

Check the LEDs is to control which cells that contain samples that will be measured.

There are 6 cells in the sample holder. The program makes it possible for

measurements to be conducted in all pockets at the same time. For example this

might be necessary and helpful if there is a need to compare the activity of two or

more catalase solutions.

Time between measurements is the time that the program waits between taking

measurements. The time is measured in seconds.

Length of experiment is how long time the experiment will be running. The program

makes it possible to follow the change in absorbance over a long period of time. The

time is measured in minutes. So the number of measurements that will be taken is

60∗𝑙𝑒𝑛𝑔𝑡 𝑕 𝑜𝑓 𝑒𝑥𝑝𝑒𝑟𝑖𝑚𝑒𝑛𝑡

𝑡𝑖𝑚𝑒 𝑏𝑒𝑡𝑤𝑒𝑒𝑛 𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑚𝑒𝑛𝑡𝑠.

20

Wavelength sets the wavelength of the light that is being beamed through the

sample.

Measurement mode sets the mode of the measurement. The two modes to choose

from are Absorbance and Transmittance.

Auto zero sets the zero level reference to the sample of choice. The absorbance or

transmittance measurements will then be compared to this level.

ON, OFF and STOP are to control the experiment measurement.

Spectrophotometer port indicates through which port the communication with the

spectrophotometer is carried out.

Real-time data shows the current data reading which updates every 500 ms when

measurements are not conducted. This is necessary when determining if the

hydrogen peroxide solution has the right absorbance value. When a measurement is

carried out the data is updated to the latest data reading during the experiment.

Graph of the measurements shows the measurements in a graph so that the results

will be easy to grasp. The graph gets updated every time a new measurement value is

taken.

Enter a description is where information about the measurement is written. It ends

up in the header of the spreadsheet file. Things that are important for the

Figure 10 The graphical user interface of the photometric mode in the program.

21

experiment, such as the date or in which quantities the solutions are, can be entered

here.

File name shows the current file name where the data is being stored.

Measurement data shows the exact values of the measurements being taken. It gets

updated after every measurement taken.

3.2.2 The flow chart of the photometric mode

In the program the tasks are running one after the other. When the program is started it

ensures that the program and machine is ready for measurements. First it clears the graph,

the table and the text field from old inputs so that the user will not be confusing old

measurements with the current one. Then it sets all the parameters to the default

parameters. Since the photometric mode was designed to do catalase solution

measurements the default wavelength will be set to 240 nm because these measurements

are carried out in this wavelength. By default the time interval will be set to 10 seconds and

the length of the experiment to 100 minutes. The sample holder is moved so that cell 1 is

positioned in front of the measurement beam.

While not making measurements or auto zeroing the program is taking an absorbance

measurement every 500 ms and displaying it on the screen. If there is front panel activity

this will get interrupted and the program will manage the other task first. It can get

interrupted by someone starting a measurement, pressing the button for an auto zero or

pressing the button for Stop.

When an auto zero is performed the program first sets the wavelength then it shows a

dialogue with information on how to perform the auto zero. First the sample is put in cell

number 1 and then the light protecting cover and the lid of the spectrophotometer has to be

closed. When pressing OK in the pop-up dialogue the auto zero measurement will be

performed.

When pressing the STOP button the program will be terminated. The graph and data will still

be shown until restarting the program again.

To easier understand the flow of the program a flow chart has been constructed to show the

relations between the different steps. In figure 11 the simplified image of the program is

shown.

22

Figure 11 The flow chart of the photometric mode. The big arrows on some of the boxes indicate communication with the instrument. An arrow going from the box specifies writing commands and an arrow going to the box specifies reading commands.

Since doing the measurement is the main feature of the photometric mode a flow chart for

just this part has been made. It can be seen in figure 12.

When starting a measurement the parameters first get written to the spectrophotometer.

Then the file dialogue, where the storage file is chosen, pops up. When the OK button in the

file dialogue is pressed the measurement starts. So it is important to have the solutions

already mixed and the lid to the spectrophotometer closed. Otherwise the first

measurement data might be misleading.

First the program saves the file header. Then it takes the data and starts a timer with the

pre-specified measurement interval. To draw the graph and write to table the data arrays

need to be rearranged. The program updates the graph and table after every measurement

taken and also saves it to the file. If an error were to occur the data up until that point would

be saved. The program waits for the timer to finish and then it takes another measurement

and do the whole procedure again.

23

When there are no more measurements to take or if someone manually turns the

measurement off the program will stop taking measurements and go back to showing the

real-time value on the screen and waiting for front panel activity. The graph and the table

will still be intact.

3.2.3 The spreadsheet file

In both modes it is possible to save the data that has been measured for later consideration

and analysis. For example the data can be processed in MATLAB as it has been for this

project. The spreadsheet files for the different modes look a bit different.

In the photometric mode there is the option to add a description in the header of the

spreadsheet file. Here a number of things can be written, for example the current number in

the measurement series, which date it was taken, the concentration of the solutions etc…

The spreadsheet file will contain the measurements of all the samples in the same file, if

more than one sample is being measured at a time. In figure 13 an example of the top of a

spreadsheet file can be seen.

Figure 12 The flow chart of doing the measurement in the photometric mode. The big arrows on some of the boxes indicate communication with the instrument. An arrow going from the box specifies writing commands and an arrow going to the box

specifies reading commands.

24

3.3 The Spectrum Mode

The spectrum mode allows sample absorbance characterization in a wavelength range

within 190nm-1100nm. This part of the program was added later in the project, to be a part

of the automatization of the UVmini-1240. It is possible to choose the wavelength range for

the spectrum and also choose between measuring the absorbance, transmittance or energy

spectrum. The scan resolution, the number of scans and the display mode are also

parameters that can be defined. The graphical interface gives the user a clearer overview of

the parameters that are defined for the test.

A baseline correction can and should be done before starting a test. This is an evident

feature in the program. The spectrum is shown in a graph and also the exact data values are

shown in a table. To save the acquired spectrum a button is pressed and a file dialogue will

appear. Here a file name is chosen and the data table is saved on the hard drive of the

controller PC onto the specified spreadsheet file. If several scans were made of the same

sample, all of them will be saved in the same spreadsheet file.

This part of the program adds a feature that the spectrophotometer did not have; the

possibility to save the data taken from the spectrum measurement. This is necessary when

post-processing is desired or when comparing results.

Figure 13 The flow chart of doing the measurement in the photometric mode.

25

3.3.1 The GUI

The graphical user interface of the spectrum mode can be seen in figure 14. Here comes an

explanation for all the parameters that can be defined:

Measurement mode sets which measurements mode to use when doing the

spectrum. The different modes to choose from are absorbance, transmittance and

energy.

Wavelength range sets between which wavelengths the spectrum will be measured.

The interval must be between 190 nm and 1100 nm.

Recording range sets the amplitude of what values that can be recorded.

Unfortunately the company who made the spectrophotometer left out the command

to set this parameter. This will be fixed as soon as the company gets back to us.

Scan speed sets the speed of the scan and therefore also the resolution of the

spectrum. The resolution depends on the scan speed and how wide the wavelength

range is. There are five different speeds ranging from very fast to very slow.

Number of scans is where it is possible to choose how many scans of the sample that

should be taken. They will be taken after one another without any stop.

Figure 14 The graphical user interface of the spectrum mode of the program.

26

Display mode is where it is decided how the spectra will be displayed if more than

one measurement is taken. The two options are overlaid and sequential. Overlaid

means that all of them will be shown in the same graph. Sequential means that they

will be shown in the graph in the order they are being measured.

Gain is only available in the energy mode. It sets the gain when energy is measured.

It is possible to have a gain between 1 and 6.

Light source is only available in the energy mode. It determines the source of the

light. The options are D2 lamp, WI lamp or optional lamp.

Write parameters to UVmini-1240 will send a command to the spectrophotometer to

set the parameters determined above.

The Spectrum is a graph that shows the spectrum visually. It will be updated after the

entire measurement is done.

Spectrum measurement data shows the measurement data numerically. It will

update after the entire measurement is done.

Spectrophotometer port sets the port through which the communication with the

spectrophotometer will be conducted.

Baseline correction takes a measurement of a blank sample to set the reference level.

When pushing Save data a file dialogue will appear and the spectrum data that has

been measured will be saved to the hard drive in the specified spreadsheet file.

START starts the measurement with the set parameters.

STOP stops the program from running.

3.3.2 The flow chart of the spectrum mode

When the program is started in the spectrum mode it makes sure that the program and

spectrophotometer are ready for measurement. First, it clears the graph and table from old

input to make sure that the user does not confuse the current measurement with an old

one. Then it moves the sample holder so that cell number 1 is positioned in front of the

measurement beam.

The program is then waiting for the front panel activity. It will respond when one of the

buttons are pressed. When this occurs it will perform the necessary task and then go back to

waiting mode again.

When pressing START the program will set the current parameters to the UVmini-1240 and

then perform a scan of the sample. It can take up to a few minutes, depending on the

resolution. After the spectrum will be shown in the graph and the exact data values will be

shown in the table. If the number of scans is more than one then the next scan will begin

after the first is finished. Depending on the display mode the spectra will either all be shown

in the graph at the same time or be shown one at a time. The table will always be update to

the last spectrum data.

27

When pressing the baseline correction button a pop-up dialogue will show with the

estimated time it will take to make the correction. This may take a few minutes. After the

baseline correction is done another pop-up dialogue will show, giving the information that

the correction is finished. The baseline correction will be performed over the wavelength

range that the user specified in the program.

If the user wants to save the acquired spectrum measurements he presses the button SAVE.

A file dialogue will then appear and it is possible to choose a suitable file name for the

spreadsheet file.

The program can be stopped from running by pressing the STOP button.

Also for the spectrum mode a flow chart has been created to make it easier to follow the

flow of the program. A simplified flow chart can be seen in figure 15.

Figure 15 The flow chart of the spectrum mode. The big arrows on some of the boxes indicate communication with the instrument. An arrow going from the box specifies writing commands and an arrow going to the box specifies reading

commands.

28

3.3.3 The spreadsheet file

In the spectrum mode the header in the spreadsheet file has information, such as, which

measurement mode that was used, and which wavelength range that the spectrum was

taken in. Also the date and time is saved in the spreadsheet file. If multiple spectrum

measurements were done then all of the data of that sample will be saved in the same file.

An example of a spectrum spreadsheet file is shown in figure 16.

3.4 The SubVIs

As described in section 2.1.2 there is a need for subVis, when making a big program in

LabView, to make the code easier to follow and more structured. The subVIs can be thought

of as functions and if a certain code is used a lot of times it is better to make it a subVI to get

rid of redundant code. A lot of subVIs are used in this program and some of them have been

used both in the photometric mode and the spectrum mode.

The most important subVIs are the readVI and writeVI. They are used for the communication

between the controller PC and the spectrophotometer. They are based on the

communication protocols showed in section 3.1. The writeVI is for protocol A and the readVI

is for protocol B and B’. They are the most essential VIs for communicating with UVmini-

1240 and are the base of the control program.

Figure 16 An example of a spreadsheet file produced by the spectrum mode.

29

3.4.1 writeVI

The writeVI has a VISA port and the command to be written to the spectrophotometer as

input and an “error out” as output. The writeVI is used to set parameters in the

spectrophotometer and also to start the measurements. There is a list of possible commands

in the UVmini-1240 manual. The writeVI is used in many of the other subVIs as well as in the

main program. In figure 17 a screen shot of the code for the writeVI can be seen.

3.4.2 readVI

The readVI has a VISA port and the command for the spectrophotometer as inputs and an

“error out” as output. The readVI is for reading the status of the spectrophotometer and to

take the measurement data. The readVI can read one or several data from the buffer in the

spectrophotometers memory. It will stop reading when there is no more data left in the

buffer. The readVI as well as the writeVI is used in most subVIs and the main program. As

screen shot of the readVI can be seen in figure 18.

Figure 17 A screen shot of the code for the writeVI.

Figure 18 A screen shot of the code for the readVI.

30

3.4.3 Other subVIs

Of course the program contains a lot more subVIs than the two mentioned above. In figure

19 a relation tree between the subVIs is shown. Here is a list of all the subVIs used in the

program.

autoZeroVI – the input parameter is the VISA port. It performs an Auto Zero; sets the

reference to the specific sample, in the spectrophotometer that is connected to the

VISA port. It has writeVI as subVI.

baselineVI - the input parameter are the VISA port and the wavelength range. The

subVI performs a baseline correction of the wavelength range from a blank sample.

This is necessary to do before spectrum measurements. It uses writeVI as subVI.

clusterDataVI – the input parameters are the number of data to be arranged and the

data array of one round of measurements. The output is an array of strings clustered

together two and two, the absorbance data and the time data. It is used to get the

data into the right format for the xy-graph.

dataPointsVI – the input parameters are the scan speed and the wavelength range.

Both the scan speed and the length of the wavelength interval decide how many data

points will be taken in the measurement. The subVI calculates the number of data

points in the measurement, which is the output.

firstSampleVI – the input parameter is the boolean array of the sample pockets. The

output is a numeric which tells which pocket the first sample is in. It is used in the

move&collectVI.

lastSampleVI – the input parameter is the boolean array of the sample pockets. The

output is a numeric which tells which pocket the last sample is in. It is used in the

move&collectVI.

move&collectVI – the input parameters are the boolean array of the sample pockets

and the VISA port. The output is two arrays with the measurement data and the

corresponding time. This subVI collects data and moves the sample holder to take

the data from every sampler one time. It uses firstSampleVI, lastSampleVI, moveToVI,

readVI and writeVI as subVIs.

moveToVI – the input parameters are the VISA port and the number of the sample

pocket. It moves the sample holder so that the beam of light goes through the

sample pocket of the input number. It is used in move&colletVI.

readBufferVI – the input parameters are the VISA port and the number of data

points. This subVI reads from the memory buffer of the spectrophotometer until all

the data points have been read. The output is an array with the absorbance at the

different wavelengths.

reorderDataVI – the input is a the array with the measurement data. It orders the

data to be the right format for showing it in a table and saving it to the spreadsheet

file.

31

sampleCountVI – the input parameter is the boolean array of the sample pockets. It

counts how many samples are being measured. It is used in clusterDataVI and

reorderDataVI.

setWavelengthVI – the input parameters are the VISA port and the wavelength. It

sets the wavelength of the measurement beam in the spectrophotometer. It uses

writeVI as subVI.

spectrumScanVI – the input parameters are the VISA port, the wavelength range and

the scan speed. It tells the spectrometer to perform a scan of the spectrum over this

wavelength interval. The scan will be saved in the memory buffer of the

spectrophotometer.

Figure 19 The VI hierarchy. GUI is the main program.

32

Chapter 4

Experiments and Results

As part of the project, as well as testing the program, measurements were made with the

program’s photometric mode and spectrum mode. In this chapter the results of these tests

and what they indicate can be seen.

4.1 Data Analysis for Catalase Activity

The software itself does not contain any post-processing analysis. After the controller

program collected the data and saved it to spreadsheet files, the analysis was done in

MATLAB. The decrease in hydrogen peroxide due to the added catalase can be fitted to an

exponential curve; 10𝑎𝑥2+𝑏𝑥+𝑐 . Because it is easier to do this in MATLAB than in LabView,

the curves were fitted to the measurement data with MATLAB. This was done to get the

approximate number of minutes it takes for the absorbance to go from 0.45 to 0.4. This

number is then used in a formula to calculate the units of enzymes per ml. The formula can

be found in chapter 2.

Also the velocity of the reaction in the beginning of the process is interesting to look at, see

figure 20. To know this velocity the parameters of the exponential curve of the absorbance

has to be known. There are jumps in the data in almost all of the measurements and the

conclusion is to, when possible, do the curve fitting on the data before the jumps,

approximately the first 10 minutes. See below how the slope in time 0 is calculated.

𝑓 𝑥 = 10𝑎𝑥2+𝑏𝑥+𝑐 ↔

𝑓 𝑥 = 𝑒𝑎𝑥2𝑙𝑛10𝑒𝑏𝑥𝑙𝑛 10𝑒𝑐𝑙𝑛 10 →

𝑑𝑓(𝑥)

𝑑𝑥= 10𝑐(2𝑎𝑥𝑙𝑛10 + 𝑏𝑙𝑛10)𝑒𝑎𝑥2𝑙𝑛10𝑒𝑏𝑥𝑙𝑛 10

→

𝑑𝑓(𝑥)

𝑑𝑥 𝑥=0

= 10𝑐𝑏𝑙𝑛10

33

The curve fitting is done by first take the logarithm of the absorbance data, log_y, and then

do a 2nd degree curve fitting to get the polynomial, pol. Then the polynomial is evaluated in

every data point, eval(pol), and the exponential curve is 10 raised to all those values,

10eval(pol).

The piece of MATLAB code that fits the data to an exponential curve and calculates the time

between absorbance 0.45 to 0.4 and the slope in time 0 looks like this:

function [curvefit, time,slope0] = curve_fitting(data_series, a, b)

x = data_series(a:b,1); y = data_series(a:b,2); log_y = log10(y); log_curvefit = polyfit(x,log_y,2) curvefit = 10.^polyval(log_curvefit, x); log_curvefit40 = log_curvefit; log_curvefit45 = log_curvefit; log_curvefit40(3) = log_curvefit40(3) - log10(0.4); log_curvefit45(3) = log_curvefit45(3) - log10(0.45); time = abs(abs(roots(log_curvefit40))-abs(roots(log_curvefit45))); slope0 = 10^log_curvefit(3)*log_curvefit(2)*log(10);

0 5 10 15 20 25 30 35 40 45 500.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

Time [min]

Absorb

ance a

t 240 n

m

Catalase activity measurement with spectrophotometer

Expontential fitting

Measurement data

Slope here?

Figure 20 The figure demonstrates where it is interesting to know the velocity of the reaction.

34

This slope can then be used to calculate the number of enzyme units per ml. The formula

from section 2.2.3.4 can be used

𝐸𝑛𝑧𝑦𝑚𝑒 𝑢𝑛𝑖𝑡𝑠 𝑝𝑒𝑟 𝑚𝑙 =3.45 𝑑𝑓

∆𝑇 0.1= 𝑑𝑓 = 1 =

34.5

∆𝑇

The equation for a slope is ∆𝑦 divided by ∆𝑥. This gives

𝑆𝑙𝑜𝑝𝑒 = ∆𝐴

∆𝑇

These two equations combined give us

𝑆𝑙𝑜𝑝𝑒 = (𝐸𝑛𝑧𝑦𝑚𝑒 𝑢𝑛𝑖𝑡𝑠 𝑝𝑒𝑟 𝑚𝑙)∆𝐴

34.5

∆𝐴 equals -0.05 in this case, since the first equation is suited for when the absorbance

decreases from 0.45 to 0.40. This gives

𝐸𝑛𝑧𝑦𝑚𝑒 𝑢𝑛𝑖𝑡𝑠 𝑝𝑒𝑟 𝑚𝑙 = 𝑆𝑙𝑜𝑝𝑒∆𝐴

34.5= −𝑆𝑙𝑜𝑝𝑒

1

0.00145

This equation will be used in section 4.2.1 to calculate the number of enzyme units per ml in

the solution, using the value of the slope in time zero.

35

4.2 Measurements in Photometric Mode

As can be seen in chapter three there are various parameters to keep in mind before doing a

measurement. Also it is possible to regulate the temperature of the cuvette holder with a

manual control device on the UVmini-1240. This is used when testing the impact of different

temperatures on the samples. Also activity measurements have been carried out along the

way to get information of the number of enzyme units per ml the catalase solution contains

to give aid to the ultrasonic testing project.

4.2.1 Changes in catalase activity due to storage temperature

The first thing that was needed to be tested for the project was how it is possible to store

the catalase solution for the measurements. This is important to know when using the same

solution for measurements that are ongoing for a long period of time. Since it is known that

temperature has an impact on the enzyme activity it is important to know how much. If the

catalase activity is changing over time and same solution will be used for the measurements

the results might be useless for internal comparison.

Measurement preparations

The two options considered for storing the catalase solution is either in the freezer or in the

fridge. This gives us the storing temperature of -18° C and 4° C respectively. A solution of 20

ml buffer with about 50-100 units of enzymes per ml was produced, and divided into two

glass jars. One jar was put in the freezer at -18° C and the other was put in the fridge at 4° C.

The buffer solution used in the experiments was also kept in the fridge at 4° C. The hydrogen

peroxide solution was produced just before doing the experiments.

Measurement procedure

The measurements were carried out regularly during 3 weeks, to get a time graph of the

activity change. The measurements were done in room temperature, with the sample holder

having a temperature of 25° C.

First do an auto zero on a blank sample (only potassium phosphate buffer), then make sure

that the hydrogen peroxide solution has an absorbance between 0.52 and 0.55.

The mixtures of solutions for the measurements in the spectrophotometer were done as

described in section 2.2.3, which is 2.9 ml of hydrogen peroxide solution and 0.1 ml of

catalase solution. These solutions were mixed in the cuvettes just before starting the

36

0 5 10 15 20 25 30 35 40 45 500.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

Time [min]

Absorb

ance a

t 240 n

m

Catalase activity measurement with spectrophotometer

Expontential fitting

Measurement data

measurement in the program. The two samples were measured at the same time using the

multiple measurement mode in the program.

The first day the catalase activity was measured before the samples went into the fridge and

freezer. The result of this measurement can be seen in figure 21. The jumps in the data are

most likely due to bubbles appearing in the reaction mixture and getting stuck on the walls

of the cuvette. To get around this problem it is possible to try to evaluate the data before

the jumps, approximately the first 10 minutes. Also, to avoid initial mixing problems it is an

alternative to consider data from minute 1 instead of time 0.

A measurement with both samples will produce a graph that looks like figure 22, which is

measurement number 7 in the measurement series.

Figure 21 The first measurement of the catalase activity for the measurement series of storage temperature. The blue dots are the measurement values and the red line is the exponentially fitted curve to the

measurement data.

37

0 10 20 30 40 50 60 70 800.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

Catalase actvitiy measurement with spectrophotometer

Absorb

ance a

t 240 n

m

Time [min]

Solution in freezer

Solution in fridge

Results

During 3 weeks 25 measurements were carried out to see the change in the catalase activity

due to storage temperature. What is being sought after is the time, in minutes, it takes for

the absorbance of the hydrogen peroxide solution to drop from 0.45 to 0.4. This number can

be used in an equation, see section 2.2.3, to calculate the amount of enzyme units per ml in

the solution. The more active the catalase solution is the shorter time it will take for the

absorbance of the hydrogen peroxide solution to drop from 0.45 to 0.4. There are two

hypothesis for the outcome of the result. One is that the activity in the catalase should drop

more in the sample that is stored in the fridge during the period of the experiment. This is

because high temperatures can denaturize enzymes. The other is that ice might damage the

structure of the catalase and that the activity goes down more in the sample in the freezer.

In figure 23 the results from the measurements can be seen. As can be seen the

measurement of the data is not very consistent, especially for the sample in the freezer.

Reasons for the inconsistency of the measurements will be discussed in chapter 5. But when

a trend line is drawn of the data it indicates that the sample in the freezer has more activity

after 22 days, se figure 23. Unfortunately the data is too scattered to draw any real

conclusions from. What can be seen is that there is no significant change in activity the first

Figure 22 The 7th

measurement of the catalase activity for the measurement series of storage temperature. The blue dots are the measurements from the solution in the freezer and the

red dots are the measurements from the solution in the fridge.

38

11 days, no matter storage temperature. This is information that can be useful for the

project.

Another way to measure the activity is to look at the velocity of the absorption curve at time

0. To calculate this we need the parameters for the exponential curve fitted to the data.

MATLAB was used to fit the exponential curve to the measurement data. In section 4.1 it is

shown how the formula for the velocity at time 0 is derived.

When the velocity at time zero of all the measurements is plotted the result will be the

graph in figure 24. The difference between the solutions in the freezer and the fridge is now

not as big. But still it is indicated that the solution in the freezer has a higher activity after 21

days.

Figure 23 The acquired data from the storage temperature measurements. The graph shows how many enzyme units per ml the solution contains. These values were obtained with the formula in

section 2.2.3.4.

0 5 10 15 20 250

10

20

30

40

50

60

Days

Enzym

e u

nits/m

l

Result from storage temperature measurements

Solution in fridge

Solution in freezer

Trend line fridge

Trend line freezer

39

0 5 10 15 20 250

10

20

30

40

50

60

Days

Enzym

e u

nits/m

l

Result from storage temperature measurements

Solution in fridge

Solution in freezer

Trend line fridge

Trend line freezer

0 10 20 30 40 50 60 70 80 90 1000.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

Time [min]

Absorb

ance a

t 240 n

m

Catalase activity measurement with spectrophotometer

Solution from freezer

Solution from fridge

Figure 24 The acquired data from the storage temperature measurements. The graph shows how many enzyme units per ml the solution contains, calculated from the reaction velocity in time zero. These values

were obtained from the formulas in section 4.1.

One last test was done on the catalase solutions 8 weeks after the first measurement was

taken. This test showed clearly that a higher activity is maintained in the sample in the

freezer than in the sample in the fridge. This can be seen in figure 25. But, as seen in figure

23 and 24, the measurements from the freezer are widely spread and what is wanted is not

only a maintenance of the activity but also results that are repetitive.

Figure 25 The catalase activity measurement of the sample in the freezer and fridge done 8 weeks after starting the experiments. It can be seen that the activity is higher in the sample from the freezer than in the

one from the fridge. Also abnormalities in the activity are visible.

40

0 5 10 15 20 25

0.4

0.45

0.5

Temperature measurement 1

Time [min]

Abs

orba

nce

at 2

40 n

m

0 5 10 15 20 25

0.4

0.45

0.5

Temperature measurement 2

Time [min]

Abs

orba

nce

at 2

40 n

m

0 5 10 15 20 25

0.4

0.45

0.5

Temperature measurement 3

Time [min]

Abs

orba

nce

at 2

40 n

m

0 5 10 15 20 25

0.4

0.45

0.5

Temperature measurement 4

Time [min]

Abs

orba

nce

at 2

40 n

m

20° C

30° C

40° C

50° C

4.2.2 Changes in catalase activity due to surrounding temperature

The second thing to be tested for the project is the impact of the surrounding temperature

of the sample while the hydrogen peroxide is reacting with the catalase. This is important

because we want the reaction to be as high as possible when doing the ultrasonic testing.

Since the catalase is working in the cells in bodies of temperature around 37° C the theory is

that these are the temperatures where the activity is the highest.

Measurement preparation

For this experiment the catalase solution needed to be diluted to slow down the reaction.

When the reaction is slowed down it is easier to compare the activity between the different

temperatures. The wanted time for the absorbance to go from 0.45 to 0.4 is around 15

minutes. The temperatures for the first measurements that were used were 20° C, 30° C, 40°

C and 50° C. The different temperatures were measured 4 times each, with the same

catalase solution.

Figure 26 The catalase activity measurement done with different surrounding temperatures. The temperatures measured were 20° C, 30° C, 40° C and 50° C. The graphs show the decrease of light absorbance at wavelength 240

nm caused by the decrease of hydrogen peroxide in the samples.

41

Measurement procedure

The procedure is the same as the storage temperature measurements. First the temperature

was regulated with the temperature wheel on the CPS controller. When putting the sample

of the hydrogen peroxide solution into the cuvette holder it is important to let it there for a

few minutes to get the right temperature. After that the catalase is added and the

measurement is started. The graphs of the measurements can be seen in figure 26.

Results

To see the general result the averages were taken for each of the temperatures and plotted

in the same graph. This can be seen in figure 27. There it can be seen that the result does not

at all go confirm with the hypothesis that the activity should be greater around 30 - 40° C. It

seems that the activity is higher when the surrounding temperature is lower. To know if the

activity would increase more when going below 20° C another measurement series was

carried out with a surrounding temperature of 10° C and 20° C. 4 measurements of each

temperature were made. An average of these 4 measurements can be seen in figure 28. No

significant difference was found between the two temperatures, so the temperature for

ultrasonic measurements was decided to room temperature (25° C).

0 5 10 15 20 250.42

0.43

0.44

0.45

0.46

0.47

0.48

0.49

0.5

0.51

0.52Results rom surrounding temperature measurements

Time [min]

Absorb

ance a

t 240 n

m

20° C

30° C

40° C

50° C

Figure 27 The graph shows the average curves for the different temperatures made from the four absorbance measurements for sample temperatures 20° C, 30° C, 40° C and 50° C.

42

4.3 Measurements in Spectrum Mode

The spectrum mode gives us information of the behaviour in a wider wavelength range.

When catalase test are analysed using this mode, the peak related to hydrogen peroxide

appears clearly and justifies the wavelength chosen for the photometric analysis. Obviously,

the spectrum measurement takes a longer time for data acquisition and cannot be used to

assess the enzymatic activity. Nevertheless, it can be used to see the general evolution of

the spectrum.

Also some spectra for preparation of HPLC measurements of sugars were made. These

measurements are related to another spectrophotometric application. In the HPLC system,

just one detection wavelegth may be used in the detector. To determine the best sensitivity,

peaks of the absorbance may be found. These peaks were obtained using the

spectrophotometric application developed in the spectrum mode.

0 5 10 15 20 250.34

0.36

0.38

0.4

0.42

0.44

0.46

0.48

0.5

0.52Average of temperature measurements, 20 and 10 degrees

Time [min]

Absorb

ance a

t 240 n

m

20° C

10° C

Figure 28 The graph shows the average curves for the different temperatures made from the four

absorbance measurements for sample temperature 10° C and 20° C.

43

4.3.1 Spectrum measurements of hydrogen peroxide

To see how the hydrolysis affects the spectrum of the hydrogen peroxide solution a few

spectrum measurements were done. First a baseline correction with the buffer solution was

done and then a spectrum of the hydrogen solution was taken before hydrolysis. The

catalase was added and two more spectra were taken. After activity stagnated another

spectrum was taken. The results can be seen in figure 29. The graph indicates that the

hydrogen peroxide is decreasing in the sample. It also shows that the hydrogen peroxide

absorbs the most light at wavelengths approximately between 200 and 250 nm. These peaks

are in agreement with the wavelength used in the absorbance measurements of catalase

activity using the photometric mode.

In figure 30 a graph, between the wavelengths 190 nm and 300 nm, of the spectrum is

shown. This is the most interesting range since it is where the peaks appear.

100 200 300 400 500 600 700 800 900 1000 1100-0.5

0

0.5

1

1.5

2

2.5Hydrogen peroxide solution during hydrolysis

Wavelength [nm]

Absorb

ance

Before hydrolysis

During hydrolysis

During hydrolysis

After hydrolysis

Figure 29 The graph of spectra measurements of hydrogen peroxide solution before, during and after

hydrolysis.

44

4.3.2 Spectrum measurements of different sugars

These measurements were conducted to see the characteristics of some different sugar

solutions before doing HPLC analysis of them. Sugars are normally analyzed using refraction

index measurements in HPLC systems. Nevertheless, these results show a UV absorption

peak at 190nm. With this wavelength HPLC measurement could be made. The

measurements were carried out for the following sugars:

Fructose

Glucose

Maltose

Saccharose

The solutions were made with distilled water and 1 % (w/w) of the sugar being tested. A

baseline correction was done first on the distilled water before the spectrum measurements.

The results can be seen in figures 31 and 32.

190 200 210 220 230 240 250 260 270 280 290 3000

0.5

1

1.5

2

Hydrogen peroxide solution during hydrolysis, zoom

Wavelength [nm]

Absorb

ance

Before hydrolysis

During hydrolysis

During hydrolysis

After hydrolysis

Figure 30 A zoomed graph of the hydrogen peroxide spectra.

45

200 400 600 800 1000-1

0

1

2

3Fructose 1%

Wavelength [nm]

Absorb

ance

200 400 600 800 1000-0.5

0

0.5

1

1.5Glucose 1%

Wavelength [nm]

Absorb

ance

200 400 600 800 1000-0.5

0

0.5

1

1.5Saccharose 1%

Wavelength [nm]

Absorb

ance

200 400 600 800 1000-0.5

0

0.5

1

1.5Maltose 1%

Wavelength [nm]

Absorb

ance

Figure 31 The spectra of the different sugars, between the wavelength 190 nm to 1100 nm.

190 195 200 205 210 215 220 225 230 235 2400

0.5

1

1.5

2

2.5Fructose 1%

Wavelength [nm]

Absorb

ance

190 195 200 205 210 215 220 225 230 235 2400

0.5

1

1.5Glucose 1%

Wavelength [nm]

Absorb

ance

190 195 200 205 210 215 220 225 230 235 2400

0.5

1

1.5Saccharose 1%

Wavelength [nm]

Absorb

ance

190 195 200 205 210 215 220 225 230 235 2400

0.5

1

1.5Maltose 1%

Wavelength [nm]

Absorb

ance

Figure 32 The zoomed graphs of the sugar spectra, in the wavelength range 190 nm to 240 nm.

46

4.4 Discussion

While doing the measurements sometimes abnormalities occurred, see figure 21. Below you

can see the discussion about this.

4.4.1 The storage temperature measurements

A series of experiments were made to see which temperature to store the catalase solution

in. Two temperatures were considered, 4° C and -18° C. A catalase solution was made and

divided into two jars and put in the freezer and fridge respectively. Then activity

measurements were carried out on the samples over the course of three weeks. The

measurements indicate that the freezer kept more activity after 3 weeks. They also indicate

that the first 11 days there is no significant difference in activity for where the solution is

being stored.

As seen in this chapter the measurements for the storage temperature are widely varying,

especially for the sample in the freezer (-18° C). There can be many reasons for this, for

example stains on the walls of the cuvettes. The most likely reason though, is that the

bubbles formed by the oxygen gas, which is a product of the reaction, are getting stuck on

the walls of the cuvette causing diffraction of the light hence altering the absorbance. This

can be prevented by preparing the insides of the cuvettes, so that the bubbles do not easily

get stuck on them. Because the results are so wide-ranging, it is hard to draw any reliable

conclusion from the measurement data.

The big variations for the sample in the freezer might be due to a non homogenous freezing

of the solution. When the catalase solution was taken from the sample, for measurements,

the sample only got to melt to the point that it was possible to take 0.1 ml of liquid. So part

of the sample was still solid. If the freezing is inhomogeneous it might explain the big

variations in enzyme activity.

4.4.2 The surrounding temperature measurements

Four tests of different temperatures were conducted to see how the surrounding

temperature affects the catalase activity. The surrounding temperature, i.e. the temperature

of the sample holder, was controlled by the CPS controller’s temperature wheel. The

temperatures that was tested were 50° C, 40° C, 30° C, 20° C and 10° C. The activity went

down when the temperature was above 30° C but there was no significant difference

between 20° C and 10° C.

As can be seen in figure 26 and 27 the activities of the different temperatures are about the

same speed in the beginning and then the higher temperatures decline. When the

47

measurements were started only the hydrogen peroxide solution, that was already sitting

the cuvette holder, had the temperature stated. The catalase solution was in room

temperature when added. Therefore the first minute of measurement might not be so

accurate when looking at temperature influence.

4.4.3 Accuracy of the curve fitting

The accuracy of the curve fitting has been evaluated by looking at the MSE (mean square

error) for the storage temperature measurements. The MSE for the sample in the fridge is

lower than for the sample in the freezer. In figure 33 a plot of the MSE can be seen. The

biggest contribution to the MSE is probably the jumps in the data due to the bubbles. The

equation for calculating the MSE is (𝑥 − 𝑥)2.

0 5 10 15 20 250

0.002

0.004

0.006

0.008

0.01

0.012

0.014

0.016

0.018

0.02

Days

Mean s

quare

err

or

Storing temperature measurements, MSE of the curve fittings

Solution in fridge

Solution in freezer

Figure 33 The MSE of the curve fitting to the storage temperature measurements.

48

Chapter 5

Conclusions

In this chapter some of the reflections and thoughts about the thesis work will be presented.

Also improvements and future work will be discussed.

5.1 The Program

The assignment was to create an interface to control the spectrophotometer UVmini-1240.

First the communication link between a controller PC and the spectrophotometer needed to

be created. A user-friendly GUI was formed on the controller PC with the graphical

programming language LabView. Two modes; the photometric mode and the spectrum

mode, was created to control the different measurements of the spectrophotometer. The

different modes will be explained more below. The program is already being used in the

department of acoustics in C.S.I.C in Madrid for enzyme activity measurements and

spectrum measurements. It is successfully executing the tasks it was developed to manage

and it is a useful tool for working with enzyme activity analysis. But as always when only

having a short period of time to develop software there is place for improvements, both in

structure and function. The program was improved little by little when new needs were

discovered. It would have been better to start with a clear, thought-through program

specification instead of adding functions to the program as they came about.

5.1.1 The photometric mode

The photometric mode was created to simplify enzyme activity measurements. It extended

the functionality of the spectrophotometer significantly by adding the ability to take

measurements over a long time period and show the results visually in a graph and

numerically in a table. The parameters of the spectrophotometer can all be set with the

program and there are also new parameters such as time interval between the

measurements and length of the experiment. The graphical user interface is easy to use and

gives the user a good overview of the parameters used in the measurement. Also all the data

collected is saved in a spreadsheet file for later consideration.

49

5.1.2 The spectrum mode

The spectrum mode was created to take samples of the absorbance over a range of

wavelengths. In the spectrum mode it is possible to do one or more spectrum scans. The

program reads the scan from the spectrophotometer’s data buffer and produces a graph of

the result and a numerical data table. The parameters that can be set in the

spectrophotometer, such as wavelength range, measurement mode, scan speed and

number of scans, can also be set through the virtual interface. A baseline correction can be

made directly in the program. This mode makes it possible to save the spectrum data on the

hard drive of the controller PC. It also gives the user a good overview of the parameters set

with the user-friendly GUI.

5.2 The Contributions

The interface that was developed for the spectrophotometer is an important improvement

of the handling of the measurement equipment. The main advantage of the program is the

possibility to make fast data acquisition, storing the data in a spreadsheet file and showing a

graph in real-time of the absorbance vs. time.

The virtual interface was used to obtain information about the enzymatic activity of catalase.

Conclusions from the data series have been drawn. For example, it can be said that the way

of storing the catalase solution, either in the freezer or the fridge, does not matter much for

the activity the first 11 days of storage. With measurements done using the virtual interface

it was also found that the catalase is most active in temperatures around 20° C. On-going

catalase activity measurements are carried out with this program for the ultrasonic project

in C.S.I.C.

5.3 Future Work

An improvement to the photometric mode could be to add post process data analysis and

also the opportunity to open and show old measurements in the program. For this project

the post-processing data analysis of the measurements have been conducted in MATLAB,

with the data taken from the files stored on the controller PC.

Also in the spectrum mode it could be useful to have some direct post-processing data

analysis. For example instant access to the peaks and valleys could be very helpful.

An improvement for the spectrum mode could be a fast spectrum scan, to take a few points

and plot it against the wavelength. At the moment a spectrum scan takes a very long time to

perform. Unfortunately, in the spectrum mode, there was a command missing in the

50

command list for how to set the measurement range of the

absorbance/energy/transmittance. This has not yet been resolved.

Since this was just the start of a project for a new way of measuring catalase enzyme activity

there are still many things to do. C.S.I.C will continue the work with the ultrasonic

measurements. The aim is to get a procedure with ultrasonic backscattering to successfully

analyze the activity of catalase, increasing the sensitivity obtained using the

spectrophotometer.

51

References

[1] Gordon G. Hammes “Enzyme Catalysis and Regulation”, Elsevier Science & Technology

Books, 1982.

[2] Yves M. Galante, Cristina Formantici, Enzyme Applications in Detergency and in

Manufacturing Industries, Current Organic Chemistry, 20030901 7(13): p.1399, 2003.

[3] Maps Group (2010, March 25), History of Enzymes [Online]. Available:

http://www.mapsenzymes.com/

[4] Eduard Buchner, Über alkoholische Gärung ohne Hefezellen, 1897.

[5] Enzymes Technical Association (2010, March 25), Enzymes in textile industry [Online].

Available: http://www.fibre2fashion.com

[6] N.E.M Business Solutions (2010, March 25), The Basics of Making Cheese [Online].

Available: http://www.cip.ukcentre.com/

[7] Nomenclature Committee of the International Union of Biochemistry (NC-IUB), Units of

Enzyme Activity, 1978.

[8] Robert Eisenthal,Michael J. Danson, “Enzyme assays: a practical approach”, Oxford

University Press, 2002.

[9] Todd MJ, Gomez J, Enzyme kinetics determined using calorimetry: a general assay for

enzyme activity?, 2001.

[10] Akio Tsuji, Masako MAEDA and Hidetoshi ARAKAWA, Chemiluminescent Enzyme

Immunoassay A Review, 1989.

[11] P. Pietta, P. Mauri and M. Pace, HPLC assay of enzymatic activitie,

Chromatographia Volume 24 Number 1 / December, 1987.

[12] Beers RF, Jr., Sizer IW. A spectrophotometric method for measuring the breakdown of

hydrogen peroxide by catalase. J Biol Chem 1952;195:133–140, 1951.

[13] Vitaly Buckin, Eugeny Kudryashov, and Breda O’Driscoll, High-resolution ultrasonic

spectroscopy for material analysis, Spectroscopy Perspectives, 2002.

[14] Sigma-Aldrich Inc, Product Information, product number C 1345.

APPENDIX A

APPENDIX A