Practical 1: Is This Blood Real? (Answers) PDF

**Practical 1: Is this blood real?** **Dr. Maggy Fostier** Aims and intended learning outcomes **Aims** To identify and quantify biological molecules (haemoglobin, proteins and DNA) using spectrophotometry. Proteins and DNA are covered in the test your understanding quiz. **Intended Learning Ou...

**Practical 1: Is this blood real?** **Dr. Maggy Fostier** Aims and intended learning outcomes **Aims** To identify and quantify biological molecules (haemoglobin, proteins and DNA) using spectrophotometry. Proteins and DNA are covered in the test your understanding quiz. **Intended Learning Outcomes (ILOs).** At the end of the practical you will be able to: **Transferable research skills** - Make calculations to prepare solutions and dilutions - Plot a calibration curve (graph) by hand or using Excel - Use a calibration curve to calculate the concentration of a solution and convert units **Specific biological techniques** - Explain how to measure and deliver small volumes of liquids accurately (automatic pipette) - Explain how to use a spectrophotometer to determine an absorption spectrum and measure absorbance at fixed wavelengths. - Identify molecules in solution based on their absorption spectra. - Plot a standard curve of absorbance versus known concentrations of haemoglobin and use it to calculate an unknown concentration [Video: Dr Fostier Introduces Practical 1](https://video.manchester.ac.uk/lectures/52ad590318cc7901adeb647567d61a12/ece69bdb-ab6e-42d0-86d5-459b809c9028/) **Useful Data-handling Learning Modules** - \'Measurements and units\' - \'Moles and concentrations\' - \'Accuracy and precision\' - - \'Functions and equations\' - \'Graphs and trendlines\' **The 6R approach** Logo, company name Description automatically generated The 6R guides to manage plastic sustainably were developed in 2019-20 by Maggy Fostier and Ruth Grady (School of Biological Sciences) with staff and students for laboratories and home/campus. **6R has been shown to have immediate impact: ** - In our pilot before COVID, we reviewed 12 practical classes with **6R for labs** and saved 37,000 plastic items YEARLY. We have since started the process of reviewing all our classes with the help of undergraduate and postgraduate students. 6R is also being rolled out to all our research laboratories to try to offset some of the estimated 5.5 millions tons of plastic generated by Biomedical and agricultural research worldwide yearly.   - In 2023, \>500 Y1 SBS student were asked to adopt **6R home and campus for a week.** On average, each student saved WEEKLY 10 items of plastic, £8 and recycled 9 extra items of plastic. Importantly 80% thought their actions could be sustained long term. We will redo this 'experiment' this year and I hope your impact will be even greater. **6R is simple to use**: the posters (under the **green corner tab** on Bb) give the broad principles and access to the full guide via the QR code. For home and campus, the [full guide](https://docs.google.com/document/d/16wjxe1q4OAjBTvzqIEfk9czKxUgdXbISYN78Dq2gmv0/edit?usp=sharing) tells you everything you need to know about managing plastics in Manchester. The guide is updated 2-3 times a year as recycling news keep evolving. Users are welcome to share 6R widely or adapt the poster and guide. It'll be great to know if you adapt it. **Also check the [videos ](https://www.youtube.com/channel/UCiONsNvprJGR4CSkPvAni0A/videos)and [text ](https://www.sustainability.manchester.ac.uk/waste/recycleweek2022/)answers to all your Qs about recycling (plastic and others). **This material was created by the UoM single use plastic group. Our social media usernames are: \@UoMSust on Twitter, Facebook and Instagram.  Besides becoming more sustainable in your own life, you can take on some volunteering to help promoting a more sustainable world. - You can volunteer to be a UoM [**6R champion**](https://find-volunteering.manchester.ac.uk/opportunity/result/37401) and help us disseminate, improve or adapt the 6R guide (4-12 hrs/semester and counting towards the [Stellify award](https://www.stellify.manchester.ac.uk/)). You may also want to contribute a blog to the quarterly [FBMH Environmental sustainability Good Newsletter](https://documents.manchester.ac.uk/display.aspx?DocID=68589). - You can become a [**[sustainability champion]**](https://www.stellify.manchester.ac.uk/stellify-award/leadership/). This is a year long - step up and lead - commitment, counting towards the [Stellify award](https://www.stellify.manchester.ac.uk/). - Contact: if you have queries/ideas. Sustainability is embedded in our curriculum - SBS offers many lecture units to help understand climate change and its impact on health, agriculture, ecology. Some units also focus on adapting to climate change and conservation.  - In your final year, you can do an Environmental Sustainability Project to help us progress. - UoM also offers UCIL units to equip our students for dealing with the global climate crisis.  **Summary of Practical 1  ** This online practical allows you to explore various applications of using a **micropipette** and a light **spectrophotometer**. In the live Lab sessions 1 and 2, you will be able to practise using both so you can become skilled in their operation. **Online** **Practical 1: Is this blood real or fake?** - Part 1: Use an automatic micropipette. - Part 2: Use spectrophotometry to **identify** proteins/molecules via their signature absorption spectrum -- applied to haemoglobin, theatrical blood and a mystery solution - Part 3: Use spectrophotometry to **quantify** proteins via their absorbance at a given wavelength -- applied to haemoglobin **The questions in green test your understanding of the theory behind a technique, how to analyse and interpret results, data handling skills**. - You are encouraged to carry these out with peers (e.g. from your tutorial group) as the discussions can help with motivation for online learning and to clarify content and understanding. - Support available: Data handling modules (all accessible at any time), data handling clinic, laboratory sessions, PASS sessions. Risks associated with this practical ==================================== If these experiments were carried out in a laboratory. As for any wet lab, wear goggles and gloves to protect your skin and eyes. For the spectrophotoscopy experiment, we use purified bovine haemoglobin (low risk associated with it being a blood product), saline solution and washable theatrical blood (no risk). PART 1. Learning to use micropipettes (automatic pipettes) ========================================================== Micropipettes are used every day in research labs for three purposes: 1. Accurately and precisely transfer volumes of liquid in the microliter range. 2. Estimating/Measuring a quantity of liquid. 3. Mixing liquids or resuspending solids (centrifugation pellets or beads). This video provides a great [guide to using a micropipette](https://www.youtube.com/embed/uEy_NGDfo_8). Watch it carefully and answer the questions below. You can also watch a shorter and fun video, part of the series: *[\'what is this thing?](https://youtu.be/g6F48qGcLiQ)\'* NB: Depending on the manufacturer, the volume range of pipettes (visible on top or the side usually) and the volume visualiser can vary (especially for the P1000). You thus need to understand how to use this information to use the equipment in the appropriate way. Below are some tasks to help. **TASK 1. Select the correct micropipette and tip**. Graphical user interface Description automatically generated Referring to the range chart above, select the micropipette *(P10, P50, P200 or P1000)* most appropriate for the volume you need and the correct tip *(blue, yellow or white)*. NB: Sometimes, two micropipettes are suitable, you can have another go, just to check. Tip: Convert volumes into ul. Refer the data handling module on 'Measurements and **units'** if unsure. Each module tutorial is accessible at all time. Hint: 1 ml is 1000 ul  **TASK 2. Setting the volume correctly.** Next, set/read the volume accurately (NB: in the quizzes, I cannot use symbols, so µl is ul) Top of Form HINT: For the P10 or P20, the precision goes to a decimal number - usually shown in a different colour or after a clear line. For the P50 or P200, the number displayed is the volume For the P1000, the display varies from model to model to pay attention. What you can rely on is that the maximum number dispayed corresponds to 1000 ul. Sometimes the maximum value displayed is 1000, sometimes it is 100. Notice that there are extra divisions on these models. The little blue arrow head is part of the volume setting and here it is well centered on the bottom number displayed. The number of divisions between units is either 5 or 10. Imagine how you would use this to set a P1000 for 751 ul, a P200 to 50.5 ul or a P20 to 2.05 ul. Now try with a range of pipettes. Which of these pipettes will measure 125 ul accurately? Tick all that apply.  C= 12.5 ul, D \>125 ul (125.1 or 125.2 depending on number of divisions between units), E: closer to126 ul Top of Form**TASK 3. How to use a micropipette correctly** It is important to use the micropipette correctly to avoid damage (i.e. liquid going inside of the pipette through the small hole in the attachment part), contamination, and for accuracy (see step by step method in the second diagram).  https://www.softchalkcloud.com/lesson/files/ysnUeG0v7dhZlm/11.png  For the question below, you may want to watch the video again (3\'24 onward) and cross-check with the summary diagrammed above. Let\'s see if you understand the common mistakes and their consequences. PART 2. Using spectrophotometry to [identify] proteins. =================================================================== In PART 2, we will use scanning spectrophotometry to determine the unique absorption spectrum (like a signature) of a) haemoglobin (Hb) and b) theatrical blood and their respective absorption maxima. Before we start, you need to understand what spectrophotometry is used for, what it is and how it works. **Spectrophotometry -- What does it do** **Spectrophotometry is used to identify compounds (qualitative) and their concentration (quantitative) based on the specific wavelength(s) of light they absorb.** It is used daily to determine the composition of biological samples and the concentration of specific molecules. It can also be used to monitor live biochemical reactions and measure enzyme activity, determine enzymatic kinetic constants, or characterise ligand-binding reactions. This [e-learning resource](https://ispri.ng/cjdQ) describes the material below with step by step explanations and animation and provide some quizzes (created by a final year e-learning project student). Do it first or after reading the introduction depending on your learning style. **Principle** - White light is made up of 3 primary colours - red, blue and green - and these combine to form all the colours of the rainbow. - Each colour in the visible spectrum corresponds to a particular wavelength of light measured in nanometres, nm (or 10^-9^ m) (Figure 1 A). - When white light strikes an object, different wavelengths (colours) may be reflected, transmitted or absorbed depending on the structure of the molecules within the object (1 A). - Light that is reflected enters the eye and we see the object as that colour (1 B). - For example, blue jeans reflect blue light and absorb green and red light. - When molecules are in a solution, one can detect the wavelengths transmitted and by deduction those absorbed (see Figure 1B).  - **Spectrophotometry is a technique used to identify a molecule by the wavelengths (UV or colour in visible light) it transmits and absorbs** (1 C).  A table with different colors Description automatically generated **Figure 1. Principles of spectrophotometry.** - **A)** Electromagnetic spectrum; the visible range of wavelengths is 340-700 nm, UV: 10-480 nm - **B)** When light passes through a solution containing the molecule(s) or interest, some wavelengths are absorbed by the molecules while others are reflected to our eyes and transmitted. - **C)** The spectrophotometer can detect the wavelengths transmitted, deduce those absorbed, and return an absorption spectrum. - The spectra of four food colorants are presented. - Note that some compounds show one peak of absorption (e.g. yellow colorant) while others show two (e.g. green colorant). - **D)** Table outlining for pure organic dies the correspondence between the colour absorbed and the one reflected. NB: this correspondence is less straightforward if a compound absorbs at several wavelengths. **Let's test your understanding.** The figure on the left presents the absorption spectrum of chlorophyll a (green) and b (red).  **Why are some molecules coloured?** The science behind this concept goes beyond this course and will not be assessed, but below are some elements to help understand light spectrophotometry. For those who want to know more (may relate to chemistry unit), here is a [simple video](https://www.youtube.com/watch?v=EJz-4ZzUA_s) and a more [complex one](https://www.youtube.com/watch?v=zAQz5J3Tpgs). We said earlier that when white light strikes an object, different wavelengths (colours) may be reflected (what our eyes receives and so the colour we perceive), transmitted or absorbed depending on the structure of the molecules within the object. At the molecular level, what matters is what is being absorbed. When a compound **absorbs** photons from ultraviolet or visible light, the outer (valence) electrons are excited and promoted from their lowest energy state, the ground state, to a higher energy level. The energy required for a particular electronic transition has a fixed value, and hence only **certain wavelength**(s) of light (photons of certain energy) will be absorbed. **This wavelength depends on the particular structure of the molecule**. In organic molecules, double bonds that are near to each other (separated only by one single bond) can *conjugate*: that is they can combine, and the electrons are delocalised (spread) over all the atoms. This lowers the energy required to promote the outer electrons; so, molecules with conjugated double bonds may be able to absorb light in both the UV and the visible range of the spectrum. This includes a multitude of biological molecules such as pigments, vitamins, nucleic acids, proteins and protein complexes, such as haemoglobin (Figure 2). The pattern of absorbance (*i.e*. the particular wavelengths of light that are absorbed by the conjugated bonds in a molecule) can help us to identify the molecule and determine its concentration in a biological extract. In this experiment, you will investigate haemoglobin during the online practical, and proteins and DNA in the post lab activity. Hb structure   **Figure 2. Structure of adult haemoglobin (Hb).** A) Hb is a tetramer made of 2 alpha and 2 beta globin chains. Each chain is a protein (colourless: absorbs in the UV range) which contains in its center a haem group which provides Hb with its red-brown colour. B) Structure of the haem group. Haem is an iron-containing compound of the porphyrin class which forms the non-protein part of haemoglobin and some other biological molecules (myoglobin, cytochromes, peroxydases, catalases, etc). The haem group is responsible for binding REVERSIBLY O~2~ in the lungs (oxy-hb) and releasing it in tissue (deoxy-hb) where it exchanges it for waste CO~2~ (carbamino-hb) which is transported back to the lungs. It can also bind IRREVERSIBLY CO (carboxyHb), which is why CO is poisonous.  Look at the double bonds in the structure of the haem groups. *How many of these are conjugated?*  **There are 13 conjugated double bonds in the haem group**. Conjugated systems of fewer than eight conjugated double bonds absorb only in the ultraviolet region and are colourless to the human eye. With every double bond added, the system absorbs photons of longer wavelength (and lower energy), and the compound ranges from yellow to red in color. Compounds that are blue or green typically do not rely on conjugated double bonds alone. Binding to metallic particles can also provide colour.  Optional: Hb roles in other cells than red blood cells. In red blood cells, hemoglobin exists as a heterotetramer of two hemoglobin α (Hba) and two hemoglobin β (Hbb) subunits. Studies have shown that macrophages, epithelial cells, and neurons, also contain hemoglobin (Rahaman and Straub 2013). Hemoglobins contain a heme-prosthetic group (Fe-protoporphyrin IX) which binds not only O2 and CO2 but also nitric oxide (NO), allowing them to also participate in redox and dioxygenase reactions (Reeder et al. 2010). In mesangial cells of the kidney, hemoglobin has been shown to act as an anti-oxidant (Nishi et al. 2008). In macrophages, Hbb acts to scavenge NO (Liu et al. 1999). In vascular endothelial cells, Hba has been shown to regulate NO release necessary for vasodilation when O2 concentrations are low (Straub et al. 2012). It is clear that hemoglobin has evolved to carry out diverse physiological functions in many different cell types. The role of hemoglobin in neurons in the CNS is still not clear but links are being investigated in relation to neurodegenerative diseases (Multiple sclerosis, Parkinson\'s, Alzheimer\'s). ***Want to know more? (optional).*** [[Why]](https://www.compoundchem.com/2014/10/28/coloursofblood/) do spiders and squids have blue blood.   Spectrophotometry - how does it work? ===================================== How does the spectrophotometer measure absorbed light? ------------------------------------------------------ The spectrophotometer, shown diagrammatically in Figure 3, is really a sophisticated version of the colorimeter, which you may have used previously.   This [video](https://youtu.be/pxC6F7bK8CU) explains what is being measured and how. Note that for most spectrophotometers, the value returned by the machine is absorbance (not transmittance). **Two types of lamp are commonly used: tungsten and deuterium**. Tungsten, used in household light bulbs, emits light in the visible range (340 - 700 nm) and can therefore be used for coloured solutions (which absorb light in the visible range). However, many biological molecules, such as DNA and most proteins, are colourless but still absorb light in the UV range.  Other molecules, such as haemoglobin, absorb light in both the visible and the UV regions of the electromagnetic spectrum. Deuterium lamps emit light in the range 200 - 340 nm and are therefore used to measure absorbance in the UV region. Typically, RNA and DNA absorbance maximum is 260 nm. For proteins, it is more complex as they vary in their composition. What absorbs UV light are their peptide bonds (absorb at 200 nm) and amino acids with aromatic rings (absorb at 280 nm), so for a given protein, the maximum absorbance wavelength varies, but when you have many proteins together, you will usually have peaks at around 200 and 280 nm. https://www.softchalkcloud.com/lesson/files/ysnUeG0v7dhZlm/Screen%20Shot%202016-04-19%20at%2014.03.23.png **How does it work?** A clear solution of the sample to be measured is placed in a small (\~ 3 mL) plastic tube called a **cuvette** (Figure 4). The cuvette is placed in the cell holder. Light, from a lamp emitting a **[fixed wavelength]** or a range of wavelengths (**[scanning spectroscopy]**), passes through a monochromator (the slit) so that only one wavelength passes through the sample at one time. The intensity of the light entering the solution (the **incident light**, I~0~) and that leaving the solution (the **transmitted light**, I~1~) is measured by the instrument; it is called the **absorbance** (Figure 4).  Figure 4. **Cuvette and definition of absorbance**. The cuvette is made of plastic, quartz or glass and is usually 1 cm wide. Absorbance, A, is the logarithm of the ratio of I~0~ and I~1~ and has no units. Absorbance is proportional to the concentration of the absorbing molecules in the solution as defined by the equation above (Beer-Lambert law). ε (pronounced *epsilon*) -- the molar absorption coefficient- is a measurement of how strongly a molecule attenuates light at a given wavelength and it is an intrinsic property of the molecule (L.mol^-1^.cm^-1^), l is the width of the cuvette (1 cm) and c the concentration (mol.L^-1^). **Let's test your understanding.** **\ ** **PART 2. Using spectrophotometry to identify proteins.** **AIM of the experiment**: To use scanning spectrophotometry to identify and distinguish proteins of similar colours. Steps: A. Sample preparation -- making solutions B. Produce the absorption spectra of bovine haemoglobin (Hb), theatrical blood and the mystery solution C. Analyse the results. - Can the spectra allow to identify with certainty which is which despite them being of very similar colour? - Can you identify one or both of these compound(s) in our mystery solution? **A. Experimental procedure. Samples preparation** Making solutions: - Saline solution (0.8% NaCl) - Haemoglobin solution (Hb 0.25 mg/mL dissolved in saline)  Other solutions provided: - Theatrical blood (in saline) - Mystery solution (in saline) Cuvette handling: - A cuvette is easily knocked over, so keep it in the rack of the spectrophotometer. - When you handle the cuvette, avoid touching the light path (see below left) as you could add grease or fingerprints which will obstruct the reading. - Typically, handle the cuvette via its top edges (see below right), but you can also touch the bottom part. Sample preparation: [Video](https://htserv.mhorn.manchester.ac.uk/mh_default_org/oaipmh-default/9ccc0158-9338-4cda-8ef0-525945272c3e/092ce71b-7299-4be1-8f22-d12bc11b1129/IMG_2080.mp4): Transferring 3 ml of Hb solution into a cuvette using the P1000. - To save plastics, we make and store solutions in [glass] containers: bottles, Universal tubes, or bijou bottles. - Cuvettes can be reused after rinsing between uses. - The volume requirement in a cuvette is only approximate - what matters is that the light path is submerged (see picture above). This means you can simply pour the liquid in the cuvette (\~2/3 up) and save micropipettes tips. **B. Experimental procedure. Produce absorption spectra with the light spectrophotometer** [Video](https://htserv.mhorn.manchester.ac.uk/mh_default_org/oaipmh-default/68c0cda1-be19-4bd2-b1eb-59acae86a4ec/4ece489d-4d50-45f6-bcb5-061bed728233/IMG_2081.mp4): Generating an absorption spectrum The video above shows how to setup and use the spectrophotometer. You will be given the instructions when you use the equipment in the laboratory. Below is a typical output when \'Read\' is pressed. ***The peak wavelengths are the signature of your molecule.*** You can zoom in to identify the wavelength (X axis) at the centre of each peak by using the screen arrows to identify the maximum absorbance and the corresponding wavelength.  **C. Results analysis.** Below are the absorption spectra of haemoglobin (Hb) (LEFT- taken from an older model of spectrophotometer and fake blood (RIGHT- taken from a touch screen model). The peak wavelengths have been highlighted for you. - For Hb, we have a broad peak from 260-273 nm (X axis), a sharp peak at 406 nm, and the the last peak indicated by the machine can be ignored. - For the fake blood, we have sharp peaks at 259 nm and 327 nm, and a broader peak at 514 nm, with possibly a shoulder around 430 nm - this means a lower peak may be hidden there. - You can also see that, for each peak, the machines gives the absorbance on the Y axis (e.g. 273 and 0.525). The absorbance is proportional to concentration as we saw in the introduction. We will come back to that in part 3. Hb and fake blood Questions: - Fake blood and Hb have the same brown-ish colour, but are their absorption spectra the same or do they allow to distinguish between the two? - Looking at the information below, can you attribute all the peaks for Hb and fake blood (made of sugar and three colorants: yellow, orange and red)?  A table with different colors Description automatically generated A\) Hb is a tetramer made of 2 alpha and 2 beta globin chains. Each chain is a protein (colourless: absorbs in the UV range) which contains in its center a haem group which provides Hb with its red-brown colour. B) Electromagnetic spectrum, including UV and visible ranges. C) The colour seen (i.e. reflected) is complementary to the one(s) absorbed. **D)** Table outlining for pure organic dies the correspondence between the colour absorbed and the one reflected. NB: this correspondence is less straightforward if a compound absorbs at several wavelengths. The mystery solution is now shown below.  - What do you think is the composition of the mystery solution? (focus on the peak wavelengths) - What do you think the scan will look like if you use **\'Hb in saline**\' as a reference sample (blank) instead of \'saline\' alone? Answers:  For Hb: - the peak at 273 nm is broad and correspond to different amino acids absorbing UV light. - the sharp peak at 406 nm comes from the haem group which gives the brown colour to our solution (between yellow and orange) The theatrical blood is made of sugar (260 nm: colourless) and three colorants: yellow (327 nm), orange (\~450 nm) and red (514 nm). The mystery solution is a mixture of Hb and fake blood as it contains the peaks of both solutions. If we had used '**\[Hb\] in saline'** as a blank, the mystery solution spectrum would have looked like the fake blood spectrum. The machine subtracts the spectrum from the blank from that of the sample End of part 2. **PART 3. Using fixed wavelength spectrophotometry to [quantify] proteins.** **AIM of the experiment**: To use fixed wavelength spectrophotometry to determine the concentration of Hb in unknown samples. Steps: A. Based on the absorption spectrum of Hb, identify its \'signature\' wavelength. B. Make solutions of Hb of known concentration (here dilutions of the original Hb solution) C. Measure their absorbance at the chosen wavelength. D. Use the data to draw a calibration curve and establish the linear relationship between absorbance and Hb concentration. E. Use the established linear relationship to calculate Hb concentration in unknown samples. **A. Identify the \'signature\' wavelength of Hb.** Usually, we select the absorption maxima for the signature and quantification (\~406 nm for Hb). What matters is to select a sharp and high peak, unique to your compound. Here, the first peak could correspond to many proteins and is not unique to Hb. **B. Make solutions of known concentration - a dilution series.** +-----------------------------------------------------------------------+ | **Reminder on dilution:** (refer to \'Moles and concentrations\' and | | 'Functions and Equations' data Handling modules for more information | | and practice). | | | | **DF (dilution factor) = total volume / initial volume of (stock) | | solution.** Here the stock solution is (Hb) in saline and it gets | | diluted in saline. | | | | *Example:* A 5 fold dilution has a DF of 5. The other way to describe | | it is 1 part of Hb solution + 4 parts of saline (1: 4, or \'one to | | four\'), or 1 part of Hb solution in 5 parts in total, (1 in 5, or | | \'one in five\'). | | | | If you want to produce 5 mL of this factor 5 dilution, you will need | | 1 part of Hb solution (total volume/DF = 5 mL/5 = 1 mL) and add 4 | | parts of saline up to 5 mL (total volume - 1 part, i.e. 5 mL - 1 mL = | | 4 mL). | | | | **DF (dilution factor) = Initial concentration | | of solute/final concentration of solute after dilution = Ci / Cf** | +-----------------------------------------------------------------------+ Hint: Rearrange the equation to work out the volume of 1 part first (i.e. the initial volume of Ribena). 1 part = Total volume / DF. Then subtract 1 part from total volume and you will find out the volume of diluent to add (here water). In Table 1, you are presented with the dilutions we need. Our stock solution is the one we prepared earlier: Hb 0.25 mg/ml. Table 1. Preparing the dilutions. Answers. Solution **A** **B** **C** **D** **E** ----------------------------------- ------------------ ------- ------- ------- ---------- Vol. of Hb solution (mL) 1 2 3 4 5 Vol. of saline (mL) 4 3 2 1 0 Dilution Factor **(Vf/Vi)** **5/1**=5 2.5 1.67 1.25  1 Concentration (mg/mL) **(Ci/DF)** **0.25/5**= 0.05 0.1 0.15  0.2 **0.25** **C.** **Experimental procedure. Measure the absorbance of known solution at a fixed wavelength.** **D. Produce the calibration curve and determine the linear relationship between absorbance ABS and Hb concentration** There is a linear relationship between absorbance (ABS) measured at **a given signature wavelength** of the solute and its concentration (in g/l, %, or M). **ABS = y \[c\].** The y value (slope or gradient of the calibration curve) is specific to the signature wavelength and the unit of the concentration used when making the calibration curve. As we saw earlier, the Beer Lambert law A= εl\[c\] describes this relationship when the concentration is expressed in M (molar) or mol/l. ε is called the molar absorption coefficient (L.mol-1.cm-1) and l is the width of the cuvette (cm). As the cuvette width is always 1 cm, we have **A= ε\[c\].** Solution **A** **B** **C** **D** **E** -------------------------------------------------------------------------------- ------- ------- ------- ------- ---------- Concentration (mg/mL)  0.05 0.1 0.15 0.2 **0.25** Absorbance **[MEASURED]** **at 406 nm with the spectrophotometer** 0.36 0.675 1.008 1.36 1.6 Using excel - plot the data from Table 1, ABS (absorbance) (Y axis) versus \[Hb\] concentration in mg/mL (X axis) - this is a scatter graph - add the trend line (line of best-fit) that includes the origin (when concentration is 0, ABS = 0) - add the equation of the line of best fit (also called **[calibration curve]** - eventhough it is a straight line) **ABS= \...\...\... \[Hb\]** - create a \'figure\' (i.e. a graph with a generic title, axes, and an adequate scale).   - your graph should look like the ones below, produced using [a different set of made-up data]. - the graph for Table 1 is displayed on the next page. [VIDEO](https://htserv.mhorn.manchester.ac.uk/mh_default_org/oaipmh-default/867c6627-27b7-42ba-99c5-c95132074264/9696b12b-119d-4f2a-bbd0-587e75a70579/Making_a_calibration_curve_in_excel___20190904_093022_17.mp4). Screencast on how to plot excel graph above. Also check the data handling module on Graphs and trendlines. NB: In class, you would have done this on graph paper by hand as well. If you get a chance, do practice that skill. The hardest part by hand is to draw the best fitting line. The fours graphs below are based on the made up data set below and formatted into a figure (title, axes, calibration curve equation, and an adequate scale). Three contain mistakes. Can you find them all? ------------------------------- ------- ------- ------- ------- ------- \[Hb\] mg/mL 0.1 0.2 0.3 0.4 0.5 Absorbance measured at 406 nm 0.212 0.428 0.704 0.956 1.326 ------------------------------- ------- ------- ------- ------- -------  Top of Form You are provided with 3 Hb solutions (F, G, H) of unknown concentration. Using your calibration curve **ABS= 6.6087 \[Hb\] in mg/mL** and the spectrophotometer in the fixed length setting, you will determine their concentration.  The tricky part here is that some solutions are very concentrated and need to be diluted for their ABS to be read. The maximum ABS accepted as accurate is 2.5 or 3 depending on the device. - Imagine: if \[Hb\]= 1 mg/ml → ABS = 6.6. - This reading is way above the spectrophotometer range, so the solution must be diluted in saline to get back within range. - Students must figure out through trial and error suitable dilutions and make good notes as the dilution factor needs to be taken into account when calculating the original concentration. - Sometimes they may have to dilute their sample twice, in which case dilutions factors must be multiplied (not added). Below are data to analyse: - These calculations are harder than the ones before. - The answers are on the next page. **Table 3. Determining the concentration of unknown Hb solutions. ** +-----------------+-----------------+-----------------+-----------------+ | Solution | **F** | **G** | **H** | +=================+=================+=================+=================+ | Vol. of Hb | ** ** | ** 2.5** | ** 1** | | solution (mL) | | | | +-----------------+-----------------+-----------------+-----------------+ | Vol. of saline | | ** 2.5** | ** 5** | | (mL) (if you | | | | | did an extra | | | | | dilution) | | | | +-----------------+-----------------+-----------------+-----------------+ | Dilution | ** 1** | ** 2** | ** 6** | | Factor. | | | | +-----------------+-----------------+-----------------+-----------------+ | **Absorbance | **0.911** | **1.7 ** | ** 1.05** | | measured** **at | | | | | 406 nm** | | | | +-----------------+-----------------+-----------------+-----------------+ | **CORRESPONDING |   |   |   | | ** \[Hb\] | | | | | concentration |   | | | | (mg/mL). | | | | | | | | | | Use the | | | | | calibration | | | | | curve | | | | | equation.  | | | | | | | | | | **ABS= 6.6087 | | | | | \[Hb in | | | | | mg/mL\]*,*** ** | | | | | *so | | | | | \[Hb\] = | | | | | ????*** | | | | +-----------------+-----------------+-----------------+-----------------+ | **ORIGINAL** co |   |   |   | | ncentration | | | | | (before | | | | | dilution) in | | | | | (mg/mL) | | | | | - **take DF | | | | | into account** | | | | +-----------------+-----------------+-----------------+-----------------+ | **ORIGINAL** co | |   |   | | ncentration | | | | | in M (mol/L). | | | | | Mr is 64 500 | | | | | (g/mol) (see | | | | | hint below) | | | | +-----------------+-----------------+-----------------+-----------------+ | **ORIGINAL** co |   |   |   | | ncentration | | | | | with most | | | | | appropriate | | | | | suffix, M, mM, | | | | | μM, or nM | | | | +-----------------+-----------------+-----------------+-----------------+ Hint: How else can you write 1 mg/mL? (Once you have transformed the unit, the rest will be easy). - 1 g/L - 1000 g/L (i.e. 1 kg/L) - 0.001 g/L (i.e. 1 mg/L) Table 3. Determining the concentration of unknown Hb solutions. **ANSWERS** +-----------------+-----------------+-----------------+-----------------+ | Solution | **F** | **G** | **H** | +=================+=================+=================+=================+ | Vol. of Hb | ** ** | ** 2.5** | ** 1** | | solution (mL) | | | | | | ** ** | | | +-----------------+-----------------+-----------------+-----------------+ | Vol. of saline | ** ** | ** 2.5** | ** 5** | | (mL) (if you | | | | | did an extra | ** ** | ** ** | ** ** | | dilution) | | | | +-----------------+-----------------+-----------------+-----------------+ | Dilution | ** 1** | ** 2** | ** 6** | | Factor. | | | | | | ** ** | ** ** | ** ** | +-----------------+-----------------+-----------------+-----------------+ | **Absorbance | ** 0.911** | **1.7 ** | ** 1.05** | | measured** **at | | | | | 406 nm** | | | | +-----------------+-----------------+-----------------+-----------------+ | **CORRESPONDING |   |   |   | | ** | | | | | \[Hb\] | 0.138 | 0.258 | 0.159 | | concentration | | | | | in cuvette | | | | | (mg/mL). Use | | | | | the calibration | | | | | curve equation. | | | | | | | | | | **ABS= 6.6087 | | | | | \[Hb in | | | | | mg/mL\]*,*** so | | | | | \[Hb\] = | | | | | ABS/6.6087 | | | | +-----------------+-----------------+-----------------+-----------------+ | **ORIGINAL** |  0.138 |  0.516 |  0.954 | | concentration | | | | | (before | | | | | dilution) in | | | | | (mg/mL) | | | | | | | | | | = \[Hb | | | | | observed\] x DF | | | | +-----------------+-----------------+-----------------+-----------------+ | **ORIGINAL** |  2.14.10^-6^ |  8.10^-6^ |  1.48.10^-5^ | | concentration | | | | | in M (mol/L). | | | | | Mr is 64 500 | | | | | (g/mol). | | | | | | | | | | Hb in mg/ml is | | | | | the same as g/l | | | | | | | | | | So \[Hb\] | | | | | (mol/l) =\[Hb\] | | | | | (g/l) / 64500 | | | | | (g/mol) | | | | +-----------------+-----------------+-----------------+-----------------+ | **ORIGINAL** |  2.14 μM |  8 μM |  14.8 μM | | concentration | | | | | with most | | | | | appropriate | | | | | suffix, M, mM, | | | | | μM, or nM (you | | | | | need to be at | | | | | ease with | | | | | units) | | | | +-----------------+-----------------+-----------------+-----------------+ **\ ** More explanations:  ------------------- **Use calibration curve equation to determine the unknown concentration in the cuvette** - **Here the linear relationship was defined with \[c\] in mg/ml (or g/l)** - Using the equation of the best fitting line we can determine ABS or \[C\] - The typical equation for a straight line is Y= a x+b ( a = slope or gradient, b= y value when x ( intercept) =0. Here y= 0 when x =0, so b =0. - **So we have ABS = y \[c\] which can be rearranged into \[c\] = ABS / y** - The equation tells us that ABS = 6.6087 \[Hb\] in mg/ml - So if \[Hb\] = 0.25 mg/ml, then ABS= 6. 6087 \* 0.25 = 1.65 - And if ABS = 2, then \[HB\] = 2/6.6087 = 0.3 mg/ml or 0.3 g/l - For F ABS= 0.911, so \[Hb\] = 0.138 mg/ml or g/l **Take into account dilution:** - For solution H, we had to dilute it by a factor 6 in order to get an absorbance reading below 3, required for this machine for accuracy. - Once diluted we obtained ABS= 1.05, so \[H cuvette\] = 1.05/6.6087 = 0.159 mg/ml - We now want to know the original concentration, so need to go back to a dilution formula. - Cf Vf = Ci Vi. Rearrange to find Ci = Cf \* Vf/Vi = Cf \* DF - So Ci = 0.159 (mg/ml) \* 6 = 0.954 mg/ml **Going from mg/ml to M** **End of Practical** - Well done! - You can go over the material in this online practical at any time. In order to register your completion of this resource, please take the quiz in Blackboard entitled \'Test your Understanding\' Quiz of Online Practical 1 for which you need to obtain at least 60%. The quiz can be taken as often as needed to reach this total. This is accessed on Bb - Lefthand menu \'Test Your Understanding\' or in the Content folder for Online Practical 1. - The ILOs of this online practical are examinable in the BIOL10401 MCQ examination in January. - If you struggle with any of the content, you can ask in the associated Lab session 1 that follows this OP1 or you can post a question on the Bb discussion site - You can also discuss these activities in PASS - Don\'t forget to complete any outstanding Data-Handling Skills Learning Modules and to attend the Data-Handling clinics if you have problems (see timetable). - You may also want to look through the Lab Session 1 link on Bb before your session. **Self-assessment** **Self assess if you have achieved the learning outcomes of this practical:** **Transferable research skills** - Make calculations to prepare solutions and dilutions - Plot a calibration curve (graph) by hand or using Excel - Use a calibration curve to calculate the concentration of a solution and convert units **Specific biological techniques** - Explain how to measure and deliver small volumes of liquids accurately (automatic pipette) - Explain how to use a spectrophotometer to determine an absorption spectrum and measure absorbance at fixed wavelengths. - Identify molecules in solution based on their absorption spectra. - Plot a standard curve of absorbance versus known concentrations of haemoglobin and use it to calculate an unknown concentration