How Would You Determine the Appropriate Wavelength at Which to Read an Unknown Solution?

Chem 125 - Experiment Two
Solution Color

Experiment 2 - Solution Colour, Absorbance, and Beer's Law

Goals of Experiment II

• Why are some solutions colored while others are colorless?
• Is at that place a pattern of color based on characteristics of the compound itself?
• How practise these solutions interact with visible light?
• What is in a solution, and how much of information technology is there?
• How do you prepare and use a calibration graph?

Questions you should acquire from this lesson and know earlier going into lab

• How does a solution collaborate with light?
• How do you make an absorbance spectrum?
• How do you make a calibration graph?
• How do you use Beer's Constabulary?

Questions you should learn in lab

• How does the solution color relate to its absorbance spectrum?
• How are concentration and absorbance related?
• How can you decide the concentration of an unknown sample?

Experiment Goal

Obtaining an Absorbance Spectrum

In obtaining an absorbance spectrum, you are getting a graph representation of how light is interacting with a solution, and how that relates to the solution colour. This interaction is very important in scientific and medical fields and that colour tin can give a lot of information. The color of a solution can give information on concentration of chemicals, how much acid is present, if a reaction has happened, or fifty-fifty if something has gone bad or not. In the post-obit pages, you will learn how obtain an absorbance spectrum for each of the samples you prepared, and use that spectrum to chronicle to why the solution is the color that it is.

Terms y'all will need to know for the experiment

Light

Color

Wavelength

Absorbance

Transmittance

Concepts you volition acquire

How does low-cal interact with a solution?

What types of light will a solution absorb or transmit?

How are absorbance and transmittance related?

How exercise absorbance and transmittance relate to the colour of the solution?

Skills you will learn

How to use a spectrophotometer

How to obtain an absorbance spectrum

How to use that absorbance spectrum to relate to solution color

Obtaining an Absorbance Spectrum

Solution Color

Picket the video below which shows how to prepare a solution.

The solution made was Cu(NO3)2 where the cation was Cu2+. You volition notice that the solid was blue, and the solution it fabricated in water, was a blue colour. Many times when you look at data in the CRC Handbook, it gives yous the colour of the SOLID form of the compound.

Friction match the following solid colors with the solution colors you would expect.

 Evidence/hide comprehension question...

Did you match the right colors?

There are many things that can change a soluiton colour. Many of them are based on the placement inside the periodic table, and how information technology is set up.

Another aspect that can effect the color of a solution is the charge of the cation. Take a look at the video beneath.

Notice that the Vanadium solutions are all different colors, just they are still cation. The difference is that the cation has a DIFFERENT accuse, which changes the electron configuration, and how light interacts with the solutions. This will be very of import for futurity experiments involving redox reactions, equally you are changing the charge of the cations. More on that afterward!

When you're in lab, expect at the solutions that yous make. Some questions you can ask yourself every bit you're making them are:

• Why do some solutions take a color, while other solutions are colorless?
• Do cations in some areas of the periodic table brandish colors while others exercise not?
• What are special most these areas that could cause changes in light interaction?
• Are in that location exceptions within the areas?
• If so, why? What's unlike?
• Why were some solutions made in h2o, while others were made in base or acid? Does that make a difference?

Obtaining an Absorbance Spectrum

Light & Solutions

About of the light that all of you lot encounter, like the light from the sun, is white light. Only did you know that the calorie-free coming from the sun is made up of many dissimilar colors?

It's time yous are introduced to Mr Roy K. Biv, the keeper of the colors!!

White light is primarily fabricated up of these seven colors, but the color is different at each wavelength.

You will see the private colors of each of these wavelengths.

Brilliant White Light!

When you wait at a solution, in that location is low-cal from all effectually interacting with information technology. This is how you lot are able to see the soluton colour.

Much of the time, you are seeing how things interact with white light. This light is the light that comes from the sun, and many light bulbs.

It is comprised of all the colors of the rainbow (ROY One thousand. BIV)!!! But what does that have to do with solution colour? The color of a solution depends on how the light (of many colors) interacts with the solution, and is a combination of colors from that rainbow!

Below shows how white low-cal would interact with a sample that you have.

1). All the colors of light in white light, hit the solution.

2). Each wavelength of light interacts with the molecules in the solution.

three). Some colors of low-cal are blocked by the molecules and Captivated, while others pass through and are Transmitted

A questions to consider:

What practise the transmitted colors stand for?

If y'all have a sample of a blueish solution, what colour low-cal would be the most transmitted?

Obtaining an Absorbance Spectrum

The Absorbance Spectrum

You've looked at what light does all at one time, but for many things, including this experiment, the low-cal is cleaved down into wavelengths. Each wavelength has its own color. When you see how each wavelength of light interacts with the solution on its own, you tin can generate an absorbance spectrum (graph) for your solution.

Each wavelength can absorb differently, below is an example of how that happens.

ane). The unlike colors of light hit the solution one at a fourth dimension.

2). Each color of light interacts with the molecules and some role of the light is Captivated, while the rest is Transmitted.

3). The amount of lite that is transmitted is different for each wavelength.

iv). The amount of lite that is Transmitted passes through the solution to the detector.

5). The machine and then tin can convert the amount of light that is Transmitted into the amount of low-cal that was blocked or Captivated by the solution.

As the light interacts with the sample, you lot tin can ask yourself.

• If i take a cherry solution, what wavelengths would transmit the about?
• What waveleghts would absorb the most?

Once the light interacts with your solution, you can graph it, much similar this spectrum of Chlorophyll A

Try this Activeness below with an absorbance spectrum

A question to think well-nigh:

• Look at the spectrum for Chlorophyll A.
• What color is clorophyll?
• Where is that color on the spectrum?
• What is the absorbance at this color?

Absorbance vs. Transmittance

Absorbance and Transmittance are related. As one goes upward, the other goes downwardly

Absorbance is the amout of low-cal that is blocked by sample molecules

Transmittance is the amout of light that passes through the solution.

Just HOW are they related?

Obtaining an Absorbance Spectrum

Laboratory Details

You lot will accept each of the solutions you already made, and create an absorbance spectrum for each ane. To practise this yous will use the spectrophotometer to select a wavelength of low-cal and measure and tape the absorbance of the sample at that wavelength.

Employ the labeled pic below to guide youself through the steps for taking an absorbance measurement.

ane). Tune the spectrophotometer to the desired wavelength

2). Load a cuvette with the solvent used in your sample

What did you use to brand your solution? Water? Acid? Base? This is your solvent, and this is what you will use to tare the spectrophotometer.

Make sure the clear sides of the cuvett are in the direction of the light.

3). Place the cuvette in the spectrophotometer, and shut it. Then tare the spectrophotometer.

This is just like taring a balance. You are telling the spectrophotometer that any lite it is measuring, should exist equal to zero absorbance.

4). Once the spectrophotometer is tared, then remove the solvent cuvette, and load a cuvette with your sample. Record the absorbance value that information technology gives y'all.

5). This procedure NEEDS to be repeated at EACH wavelength.

Absorbance is different for EVERY chemical at EVERY wavelength, so every time you change 1 of them, the spectrophotometer will need to be tared

Once you have your information all recorded, then you can plot each of your absorbance spectra

Y-axis will be the absorbance values

10-axis volition exist the wavelength

Look at the absorbance spectra that you create.

- Expect at the maximum absorbance in your graph

- Expect at the minimum absorbance in your graph

- What colour are your solutions?

 Show/hide comprehension question...

Experiment Goal

Generating and Using a Calibration Graph

Calibration Graphs are used to determine many things. In this experiment yous are using a calibration graph to chronicle absorbance and concentration. They are used in many fields to relate a quantity with something you can physically measure. In the lab, you will prepare standards of a known concnetration, and plot a calibration graph. You will then use that graph to figure out the concentration of an unknown sample.

Terms you will need to know for the experiment

Dillution

Linear

Straight Line

Line of Best Fit

Absorbance

Concentration

Unknown

Concepts you will learn

What is a dillution gene?

What is a line of best fit?

What is a calibration graph and how practice y'all use it?

Skills y'all will acquire

How to dillute samples

How to generate and plot a scale graph

How to work with the calibration graph to be able to decide an unknown

Generating and Using a Calibration Graph

Beer's Law

In the example of a calibration graph for this experiment, yous are plotting absorbance vs. concentration, every bit opposed to an absorbance spectrum where you are plotting absorbance vs. wavelength. But how are wavelength and concentration related to absorbance? They are all related in through the Beer-Lambert Law.

The main absorbance equation is the Beer-Lambert Law which is:

Where A is the absorbance

ε is the molar absorptivity constant . This is different for every chemical, and at every wavelength

l is the path length , the distance of solution that the light has to travel through

c is the concentration of the solution

The absorbance is based primarily on those three factors

Tooth Absorptivity Constant

The Molar Absorptivity Abiding is specific for every single solution, and at every wavelength. When y'all are taking an absorbance spectrum, and measuring the absorbance at different wavelengths, this is the only gene that is changing, as the concentration of the solution remains the same, so does the pathlength. The path length of each vial is the aforementioned, and the concentration of each of these solutions is the same, however the solutions are all different colors and volition therefore absorb very differently. The only difference to alter the absorbance, is the Molar Absorptivity Constant.

Questions to think about:

- Would the absorbance change if you use a dissimilar solvent?

- Which of the three factors would be affected past the change in solvent?

Path Length

The path length besides affects absorbance. With a longer path length, the light has to travel through more than solution, and can hit more molecules, and be captivated. This would make the aborbance increase and accept less lite transmit through makeing the solution appear darker.

The pictures below bear witness how solutions announced when you wait through a longer pathlength.

The image to the left shows a container with iii compartments. Each compartment has the same solution simply filled to different levels. When looking through the solutions horizontally, they all announced the same color because the path length is the same for all three. However, when yous wait at the image to the right, where the view is from the top, you tin meet that the solutions get darker as you lot motion from left to right. The pathlength is increasing. There are more molecules in the way of the low-cal, and then more light can be absorbed and less will exist transmitted to your centre.

This can likewise be seen in absorbance spectra, where equally the pathlength is increased, the absorbance is also increased.

Using this information, and the spectra in the effigy, see how the path length would outcome the absorbance in the post-obit questions.

Some of import information is that path length is rarely ever changed away from ane.0 cm.

Concentration

The terminal component of Beer'southward Law, is concentration. Concentration effects the absorbance very similarly to path length. If the concentration of solution is increased, then at that place are more molecules for the lite to hit when it passes through.

As the concentration increases, there are more molecules in the solution, and more calorie-free is blocked. This causes the solution to get darker considering less low-cal can get through.

Generating and Using a Calibration Graph

The Calibration Graph

When you take an absorbance spectrum, you are looking at the absorbance based on wavelength. Simply when making a calibration graph you are looking at the absorbance based on concentration.

The two plots above are an Absorbance spectrum on the left, and a calibration plot on the left.

Both are plotting absorbance, but the spectrum plots it vs. wavelength (tooth absorptivity constant) and the calibration plot is vs. concentration. When yous take an absorbanc spectrum, the molar absorptivity constant is irresolute up and down and all around, just when you are working with a calibration plot, and are merely changing the concentration, information technology shows a linear relationship between absorbance and concentration.

 IMPORTANT NOTE!!!!!!

Beer's Constabulary Merely is linear at LOW concentrations!

If the absorbance of your sample is above i.0 you will demand to dillute your sample in order to lower the absorbance

Y'all cannot extrapolate your calibration line, because there is a deviation from a straight line at higher concentrations.

Having trouble with direct lines? Not to worry, here are some very helpfull sites to help yous out! :)

- For information on "The Equation of a Straight Line"

- For information on "Best Fit Line"

- For information on "Scatter Plot with Fitted Regression Line (Excel)" 2003 Format

Generating and Using a Calibration Graph

Now You lot Try!

Now try opening a plotting progam and try making a calibration plot of your own. Please use the following values.

The Wavelength used for this calibration graph was 410nm REMEBER THIS!!!

Brand sure to include the following whenever y'all make your calibration graphs!

ALWAYS make sure they are labeled!

- Title (Include wavelength used)

- Centrality

- Centrality Titles

- Line of All-time Fit

- Equation of the Line of Best Fit

Hint for plotting a line of best fit!

Brand certain to FORCE your best fit line to go through the origin

Beer's Police has an intercept through the origin, and so your best fit line should reflect that.

Another way to find the slope of a line, when you do not accept a plumbing fixtures program bachelor, on a test for example, would be to summate the slope. This would give you a less accurate slope, but would still be acceptable when a fitting program is not available.

IMPORTANT Note!!! On Whatsoever Study or Exam, Ever show your piece of work when you preform any calculation!

Generating and Using a Scale Graph

How to Work with Plots

Y'all take two dissimilar plots, your absorbance spectrum on the left, and your scale plot on the right. The calibration plot is like taking a vertical piece through the all the absorbance spectra at the specific wavelength 410nm. The wavelength 410nm was a very good choice for the calibration plot, but how do you know which wavelengt is the best wavelength, based on the absorbance spectrum?

Choosing Your Wavelength

Wait at the images above. The left is an absorbance spectrum of 0.13mM plastocyanin, while on the correct is a calibration plot at two wavelengths.

Is the slope of the calibration line at 550nm greater than, less than, or equal to the slope at 600nm?

You tin cull whatever wavelength to create a calibration plot, the just differerence will exist the slope of the line.

When you actually choose your wavelength to create your scale graph, y'all would generally like to choose a wavelength where there is room for the concentration to decrease. Look at the spectrum above. Do you think 450nm would be a skilful wavelength to use for a calibration graph? Y'all would not choose that wavelength because when you lot lower the concentration, y'all would non exist able to see much of a difference in the absorbance, and the calculations would be inaccurate. You would most likely desire to choose wavelengths like 600nm or 250nm where there is a lot of room for absorbance change.

Generating and Using a Calibration Graph

Using your Calibration Graph!

Now for the fun part! Using the calibration plot that You lot fabricated from the data 2 pages agone. We are going to determing the concentration of an unknown solution. Make sure yous have your plot fix, because hither we go!

Here's a typical problem. Y'all take 3mL of your unknown sample and 7mL water and mix them together. The dilluted sample gives an absorbance of 0.432. What is the concentration of the initial unknown?

Where exercise you begin?! Well, you accept your calibration graph, and information technology SHOULD wait something like this, all properly labeled.

one). You have an absorbance, and you accept a straight line equation that relates absorbance to concentration. This is the line of best fit through your information.

2). At present this is the absorbance of your DILUTED solution. Just what was the concentration of your ORIGINAL solution? Recall you lot dilluted information technology one time, so you can employ the Dilution Equation

 Ready to try one on your own?

Need some practice? Here are a few more issues. Endeavour to figure them out on your own!

Mutual Errors In Scale Plots

• Spectrophotomer is not calibrated
• Abs readings are incorrect
• Diluted samples are prepared incorrectly or contaminated
• Inappropriate wavelength chosen for calibration graph
• The calibration line is not a "best fit" line

Final Thoughts

READ THE DIRECTIONS!!

You're Done! See y'all next experiment!

Questions?

For questions please attend the function hours of the GSI's or contact Nancy Kerner

nkerner(at)umich.edu

gilmorelankincte02.blogspot.com

Source: http://websites.umich.edu/~chem125/softchalk/Exp2_Final_2/Exp2_Final_2_print.html

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