Measurement Science Lab: Introduction

People Involved

Course Co-ordinator:		Dr. Joshua Edel		joshua.edel@imperial.ac.uk
		Dr. Kristelle Bougot-Robin		k.bougot-robin@imperial.ac.uk
Demonstrators:		Silvia Di-Lecce		silvia.di-lecce12@imperial.ac.uk
		Raquel Fraccari		r.fraccari12@imperial.ac.uk
		Markéta Kubánková		m.kubankova13@imperial.ac.uk
		Ali Magness		alastair.magness12@imperial.ac.uk
		Hugh Sowley		hugh.sowley12@imperial.ac.uk
Technicians:		Simon Bastians		s.bastians@imperial.ac.uk
		DeeJay Kristnah		d.kristnah@imperial.ac.uk
		Simon Turner		s.t.turner@imperial.ac.uk

Safety

The lab course is designed to be an enjoyable experience however as with any lab course safety is the number one priority and there are some simple safety rules:

1. YOU MUST WEAR A LAB COAT AND GOOGLES AT ALL TIMES IN THE LABORATORY.

1. YOU MUST NOT EAT OR DRINK IN THE LABORATORY OR BRING ANY FOOD IN WITH YOU.

1. YOU MUST NOT USE YOUR MOBILE PHONE IN THE LABORATORY.

In addition to these rules you must refresh yourself of all the safety regulations from the Foundation Laboratory Course and complete a risk assessment before carrying out any practical work.

Write Up

The Wiki Format

Since everyone is used to using Microsoft Word, why do we use a Wiki for this course? Well, the Wiki format has several advantages.

1. A full revision and fully dated history across sessions is kept (Word only keeps this during a session). This is more suited for laboratory work, where you indeed might need to go back to a particular day and experiment to check your notes.
2. The Wiki allows you to include "zoomable" graphics in the form of SVG (which Gaussview generates), and access to the 17-million large WikiCommons image library, as well as access to the Wikipedia InterWiki.
3. The template concept allows pre-formated entry. There are lots of powerful chemical templates available.
4. Autonumbered referencing, and particularly cross-referencing, is actually easier than using Word.
5. You (and the graders) can access your report anywhere online, it is not held on a local hard drive which you may not have immediate access to.
6. It has automatic date and identity stamps for ALL components.
7. And finally, Wiki is an example of a MarkDown language, one designed to facilitate writing using an easy-to-read, easy-to-write plain text format (with the option of converting it to structurally valid XHTML).

Creating a Page

1. In the address box, type something like wiki.ch.ic.ac.uk/wiki/index.php?title=MSL:XYZ1234
2. The characters MSL indicate a report associated with the Measurement Science Lab and XYZ1234 is your secret password for the report. It can be any length, but do not make it too long! It should then tell you there is no text in this page. If not, try another more unique password. You should now click on the edit this page link to start. Use a different address for each module of the course you are submitting.
3. It is a good idea to add a bookmark to this page, so that you can go back to it quickly.

Editing a Wiki

An introductory tutorial is available which complements the information here.

• A cheatsheet summarises the commands with a playpen for playing. You can write your report by simply typing the appropriate text as shown in the cheatsheet, or by using the WikEd buttons in Word-style composition.

• 

  The editing environment

  You will need to create a separate report page on this Wiki for each module of the course. Keep its location private (i.e. do not share the URL with others).
• The WikED toolbar along the top of the page has a number of tools for:
  • adding citation references,
  • superscript and subscripting (the H₂O WikEd symbol will automatically do this for a formula),
  • creating tables
  • adding links (Wiki links are internal, External links do what they say on the tin)
    1. local to the wiki, as [[mod:writeup|text of link]]
    2. remote, as [http://www.webelements.com/ text of link]
    3. Interwiki, as [[w:Mauveine|Mauveine]]
    4. DOI links are invoked using the DOI template {{DOI|..the doi string ..}} or the more modern form [[doi:..the dpi string..]]
    5. Links to an Acrobat file you have previously uploaded to the Wiki can be invoked using this template: {{Pdf|tables_for_group_theory.pdf|...description of link ...}}
    6. There are lots of other templates to make your life easier such as the ChemBox
  • If you need some help, invoke it from the left hand side of this page.
• Upload all graphics files also with unique names (so that they do not conflict with other people's names). If you are asked to replace an image, REFUSE since you are likely to be over-writing someone else's image!
  • Invoke such an uploaded file as [[image:nameoffile.jpg|right|200px|Caption]]
  • We support WikiComons, whereby images from the content (of ~10 million files) from Wikimedia Commons Library can be referenced for your own document. If there is a name conflict, then the local version will be used before the Wiki Commons one.
    • To find a file, go to Commons
    • Find the file you want using the search facility
    • Invoke the top menu, use this file in a Wiki, and copy the string it gives you into your Wiki page
    • [[File:Armstrong Edward centric benzene.jpg|thumb|Armstrong Edward centric benzene]]
• Colour can be added (sparingly) using this text fontcolor

template.  (invoked as {{fontcolor1|yellow|black|text fontcolor}} )

• Save and preview constantly (this makes a new version, which you can always revert to). It goes without saying that you should not reference this page from any other page, or indeed tell anyone else its name.
• Important: Every 1-2 hours, you might also want to make a backup of your report. This is particularly important when adding Jmol material, since any error in the pasted code can result in XML errors. The current Wiki version does not flag these errors properly, but instead just hangs the page. Whilst you can try to repair the page as described below, it is much safer to also have a backup!
• You should get into the habit of recording results, and appropriate discussion, soon after they are available, in the manner of a laboratory note book.

Assessment

This lab has been designed so that you can improve your measurement and data handling skills whilst at the same time understanding that instruments such as a UV-Vis spectrometer should not be treated as a black box. As such no formal mark will be given but rather the groups will be judged based on spectrometer design, discussions with assessor, and quality of experimental data. The winning teams will receive a departmental certificate and vouchers.

Course Materials

The exact setup for this experiment is left up to you, we have provided some guidelines on this wiki and in the lab manuals, and other downloadable materials.

Equipment

We have provided you with a kit that contains all the components that you might need to build a spectrometer, you can use as much or as little of this equipment as you like. If there is something that you feel would be beneficial we may be able to procure it for you if you can justify using the item.

Your kit contains a:

• selection of Lego
• Raspberry Pi with an Adafruit ADC connected

• black out cloth
• pair of slits
• diffraction grating (period 0.1 mm)
• broadband light source
• photodiode
• multimeter
• protractor
• keyboard, monitor, mouse

Downloads

We have created a range of materials for you to use:

Lab Manual	Download	View
Raspberry Pi Manual	Download	View
iPython Notebook Analysis	Download	View
iPython Notebook Data Acquisition	Download	View

Please supplement the information we have provided with your own research.

The Brief

One of the most commonly used measurement tools in the physical and biological sciences is a UV-Vis spectrometer. They can be used for both quantitative and qualitative analysis of samples, and to follow reactions in real-time recording kinetic data. In this lab course you will build your own spectrometer that must be able to:

1. Record the absorbance spectrum for a range of concentrations;
2. Record the angular dependence of absorbance (i.e. absorbance spectra)
3. Understand and determine measurement errors and limitations
4. Use the spectrometer to monitor kinetics.

To build your spectrometer you will be supplied with a photodiode detector, a black out cloth, an LED light source, a multi-meter, a power source, slits, a diffraction grating, a protractor, a Raspberry Pi computer, and lots of LEGO! You can use as many or as few of these items as you like, you are also welcome to make additions to the kit. In some circumstance we may even be able to supply additional parts at your request if you can justify their use.

Any measurement including those performed on your Lego spectrometer will have analytical limitations. To maximize the useful information from you measurements it is important understand both instrumental parameters to minimize errors and to use appropriate statistical methods to treat your data. This lab is designed to give you a better understanding of these aspects of collecting spectral data.

A few important points:

1. This lab course is about problem solving and therefore the lab script is not traditional and does not give you a list of steps to follow. Instead it gives an overview of the key parts of a spectrometer and the main points you need to think about in your design.
2. The experiment is run in groups of 3 – 4 and it is recommended that tasks be allocated to each member to ensure efficient progress.
3. To aid your understanding of some of the material you will need to consult other resources (e.g. demonstrators, textbooks, papers, course notes, the internet etc) and we encourage you to do so.
4. Your lab notes and the formal write-up (pseudo lab report) will be in the form of a wiki. It will be important to work on this as you go along.
5. Although there are 5 parts to this experiment, if time limited, the focus should be on the quality of the results rather than aiming to complete all components.
6. All data analysis should be performed during lab hours. A number of resources including the computer room can be used for this.

Goals of this Lab

1. Understanding the working principles of a UV-Vis spectrometer.
2. Improve data handling and data processing by taking into account instrumental and sample limitations.
3. Improve decision making especially when it comes to optimisation.
4. Improve problem solving ability.
5. Finally, have some fun when designing and optimizing the LEGO spectrometers.

Experimental Objectives and Timetable

The experiment is divided into four parts:

1. Building of a UV-Vis spectrometer using a combination of optical components, computer acquisition hardware, and LEGO!
2. How low can you go? Optimisation of the sensitivity and maximising the signal to noise using the dye Methylene Blue.
3. Recording of a complete UV-Vis Spectrum of Methylene Blue. Results to be compared using a commercial instrument.
4. Following a reaction to explore the kinetics of the reduction of methylene blue to leucomethylene blue.

Proposed Timetable

This lab is designed as a problem solving exercise and will run for 2 weeks. There is only one experiment in this lab that is to build and test a spectrometer based on the information held in this lab manual. The lab is designed to take the full 2 weeks and we recommend that you plan you time carefully a proposed time plan could be:

	Monday	Tuesday	Thursday	Friday
Week 1	Initial Spectrometer Design	Build and Improve Initial Design	Perform Concentration Studies	Optimise Spectrometer
Week 2	Perform concentration studies	Generate Absorbance spectra	Comparison with commercial instrument	Present final design

Experimental

Part 1: Designing and building a UV-Vis spectrometer

All spectrometers have five basic components, a light source, a monochromator or diffraction grating, entrance and exit slits, a detector and a device to read the detected signal. Before you begin building your spectrometer it is important that you design your spectrometer to make sure it will fulfil the brief’s requirements. This lab course is designed as a problem solving activity so this manual will not tell you how to build a spectrometer but will give you guidance and advice so that your spectrometer is as effective as possible. When in doubt ask a demonstrator – they are there to help problem solve. The Lego is for you to build the structure of your spectrometer the spatial design of your spectrometer is up to you. However, remember that it must be able to record a spectrum as a function of angle.

A schematic of a spectrometer

A schematic of a possible configuration for your spectrometer is shown in Figure 1. Key components are as follows:

1. White light source: LED’s are popular options due to their broad spectral range which typically emit between 400-800 nm.
2. Lens: Used for collimating the light and focussing the light on to the sample. Although the location and distance from the light source should be optimized, a good working range is between 4-7 cm.
3. Entrance and Exit Slits: Minimizes scattering and stray light from reaching the detector. These can be mounted directly before and after the sample holder. Templates for both the entrance and exit slits are given.
4. Diffraction grating: The grating splits the white light into individual wavelengths. See below for more details. The grating should be placed near the exit slit.
5. Detector: The photodiode detector will need to be mounted on either a pivot or swivel arm as the signal response from individual wavelengths will need to be recorded (variation of the angle $\theta$ from the grating normal). Although the position of the detector will need to be optimized to maximize the signal response, a reasonable working distance will be between 5-5 cm. A protractor can also be integrated into the spectrometer to convert angle of the detector arm to wavelength.
6. Recording: The detector records its signal as a voltage and will range between 0 and 5 V. Therefore, the detector output can be recorded on either a multimeter or for improved performance on an analogue to digital converter coupled to the raspberry Pi computer. It is recommended that initially the spectrometer is built and tested using a multimeter. Once optimized simultaneous multimeter and computer acquisition can be used.

The Beer-Lambert Law

The photospectrometer that you create must be able to approximate the extinction co-efficient for a range of substances. This is a simple process that can be calculated from the gradient of a plot of absorbance against concentration by applying the Beer-Lambert Law:

$A=\epsilon cl$

Where $A$ is the absorbance, $\epsilon$ is the extinction co-efficient,$c$ is the concentration and $l$ is the path length. A plot of absorbance against concentration will give a straight-line which can be fit to $y=mx$ where $m=\epsilon l$can be obtained. The path length is the size of your cuvette and so the extinction co-efficient is readily found. The Beer-Lambert law is not applicable at all concentrations since the absorbance will plateau at some concentration rather than continue to infinity. The absorbance is related to the intensity of light:

$A=-{\log }_{10}\left|\frac{I}{I_0}\right|={\log }_{10}\left|\frac{I_0-I_B}{I-I_B}\right|$

The intensity of the light, $I$, is normalised to the intensity recorded for the solvent (or when the concentration is zero), $I_0$. The photodiode will produce some voltage even in the dark this is known as the dark count, $I_d$, and should be subtracted from each reading. The photodiode converts intensity or photon count into a voltage and therefore can be universally replaced with for voltage to give:

$A={\log }_{10}\left|\frac{V_0-V_B}{V-V_B}\right|$

The light source that you are using is a broadband source that emits white light this light is made up of a spectrum. As the light passes through the sample one particular frequency of light will be absorbed as it provides the required energy for an electronic transition. The light absorbed by solutions of a particular colour can be found from the chemistry colour wheel.

Diffraction Grating

In a spectrometer the component that selects wavelength is known as a monochromator, the monochromator you will be using a diffraction grating as a monochromator. This delivers a particular wavelength, $\lambda$, based on the angle of the light passing from the grating, $\theta$, and the periodicity of the diffraction grating, $d$.

$d\sin {\theta }_m=m\lambda$

In this case $\pm m$ since the sine function is odd the $\pm$ symbol can be dropped and $m=1$, so the wavelength that will be transmitted after light has passed through your diffraction grating is $\lambda =d\sin \theta$. So if we had a diffraction grating with a period of 1000 nm and were interested in a violet solution we would want our photodiode to be at an angle of 35$o$ relative to the sample so that it would be detecting the change in yellow light ( 570-590 nm). You must look at the colour of the solution you are testing then position your diode at the optimal angle. This means that your diode must be fixed to a moving arm that is capable of sweeping across a range of angles something that is also vital for task 2. Using this information you should design a spectrometer, remember that science is an iterative process, so you should design, build, test and improve your spectrometer to achieve the optimal results.

Recording Voltage

To turn your spectrometer into a useful device you must connect the photodiode to something that is able to record voltage. In the first instance you should use a multimeter and record the voltage from your photodiode and then analyse the data to produce plots and fits. An iPython Notebook for this course is available from the wiki, however you are free to use any program you like. After you have completed your tests think about how to improve your spectrometer. Once you have a design that you are happy with and that satisfies the brief then you can upgrade your multimeter to a Raspberry Pi and use this to record your voltage and analyse the data on the fly. Saturation of the detector occurs at 5V therefore, readings should be below this value. Note: the Raspberry Pi must be turned on when recording signals from the photodiode even if only using the multimeter.

Using a Multimeter

Connect the red input to the red diode output and black input to the black diode output. Make sure the multi-meter is set to mV and then record the voltages on the screen in your lab book.

Using a Raspberry Pi

File:Raspberry Pi B+ top.jpg

The Raspberry Pi B⁺ model

Once you are at the stage where you are ready to use a Raspberry Pi please follow these instructions on how to use and setup a Raspberry Pi.

Part 2:Determining the useful concentration range using Methylene Blue

The majority of analytical methods follow the instrument response curve as shown in Figure 2. As part of this exercise such a curve should be generated for Methylene Blue with your spectrometer.

1. A useful starting concentration will be 0.3 mM.
2. It will be important to minimize the detection limits and modify the instrument accordingly in order to do this. Therefore, you will want to characterize the LOD, LOQ, and LOL.
3. Statistical methods should be used to quantify the detection limits. For example, you will want to make note and approximate measurement errors such as listing systematic and random errors, spread of data, mean, standard deviation, sources of noise, and finally averaging and signal to noise.
4. The dynamic range should be defined and fit to a linear model.
5. How does your detection limit compare to results obtained on a commercial instrument? Commercial UV-Vis spectrometers are available in the lab to assess this aspect. Ask a demonstrator to show you how to use them.

Schematic of an instrument response curve. LOD = limit of detection, LOQ = Limit of quantitative measurement, LOL = Limit of linearity.

Part 3: Determine the concentration of the unknown.

A sample of methylene blue with unknown concentration has been found in the lab. An aliquot of this has been given to you to quantify the concentration. Using the information obtained from Part 2, calculate the concentration.

Part 4: Obtain Absorbance spectra of Methylene Blue and compare with commercial instrument

Full Absorbance spectra of methylene blue should be obtained at three concentrations. This will then need to be compared to a commercial UV-Vis spectrometer and the following questions will need to be answered. You might have to optimize your methylene blue concentration and slit size accordingly to take into account the saturation of the detector.

1. How does the signal and noise compare between your spectrometer and a commercial instrument?
2. How do the signal intensities compare?
3. How can your spectrometer be improved?
4. Are there any obvious systematic or random errors?

Part 5: Kinetics associated with mixing methylene blue with ascorbic acid

You will study the reduction of methylene blue (MB⁺) by ascorbic acid (A) to its colourless form leucomethylene blue. More specifically you will investigate the dependence of the rate law on the ascorbic concentration acid.

In all cases you will consider, [MB⁺] to be less than 1% of [A], thus ensuring pseudo first-order conditions. A proposed rate law for this reaction is:

$-\frac{\mathrm{d}[A]}{\mathrm{d}t}=k_0[A][M{B}^{+}]=k_{\mathrm{e}\mathrm{x}\mathrm{p}}[M{B}^{+}]$

Using your spectrometer you should design an experiment to:

1. Find the rate constant $k_{\mathrm{e}\mathrm{x}\mathrm{p}}$ for the reduction of [MB⁺] with [A] = 0.045M. In terms of kinetics explain the order of the reaction and comment on its half-life. Discuss your experimental error (it will be helpful to perform repeat measurements). Not more than 3 concentrations are needed.
2. Determine the rate constant, $k_0$, by varying the concentration of [A] and keeping [MB⁺] constant. A potential useful concentration range for [A] would be between 0.002 to 0.045 M. Hint: what you measure will always be the experimental rate constant, you will therefore need to look into methods on how to obtain $k_0$ from this data set.
3. How does the rate constant compare to literature?
4. What are the statistical errors in your obtained results?