1. Intro to Spectrophotometry
Solutions & Dilutions
Intro to Spectrophotometry

2. Solutions
Solution: a homogenous mixture of two or more substances in the same phase.
Solute: substance dissolved in another substance (the solvent)
Solvent: substance that dissolves a solute, resulting in a solution
Water is the “universal solvent”
Other common lab solvents: Ethanol, Methanol, and Acetone
A textbook definition of a solution is a homogenous mixture of two or more substances.
These mixtures can be in any form of matter, however for this class we are going to focus exclusively on liquid solutions.
Solution is composed of a solute & a solvent
Solute: substance dissolved in another substance (the solvent)
Solvent: substance that dissolves a solute, resulting in a solution
*** Water is the “universal solvent” – dissolves more substances than any other liquid
Solubility is a chemical property referring to the ability for a given substance, the solute, to dissolve in a solvent.
It is measured in terms of the maximum amount of solute dissolved in a solvent at equilibrium. The resulting solution is called a saturated solution.
In a laboratory setting, solutions are an essential part of research.
Solutions are everywhere! In the lab: buffers, reagents, reaction mixtures, liquid acids and bases are all examples of solutions commonly used in the lab.

3. Solubility Solubility:
the ability of one compound to dissolve in another; maximum amount of solute capable of being dissolved by a solvent
Changes with temperature, pressure, and contamination
Listed in MSDS
The solubility of a solute can be measured by carefully weighing the solute as it is added to a certain volume of solvent (e.g. water) and stirring.
When there is a small amount of solute left which cannot be dissolved, the solution is saturated.
By calculating the exact mass of solute that has been dissolved, the solubility is determined.
Salt (sodium chloride) has a solubility of 35 g per 100g of water. So adding 40g of salt to 100g of water will leave 5g of salt undissolved.
In this lab, temperature will be the most important factor affecting the solubility, or maximum quantity of solute that can dissolve in a specific volume of solvent (see next slide)

4. Solubility Typically, for liquids:
Increased heat = Increased quantity of dissolved solute
Think of dissolving sugar (solute) in hot tea vs. ice tea (solvents)
In this lab, temperature will be the most important factor affecting the solubility, or maximum quantity of solute that can dissolve in a specific volume of solvent
Photos mined from Google Image Search

5. Concentration
Concentration: the quantity of solute present per unit volume of the solvent
When preparing a solution, you can create different strengths or concentrations for your mixture based on the amounts of solute & solvent used.
Concentration: the quantity of solute present per unit volume of the solvent
You can create:
“Dilute” solutions: add more solvent or reduce solute
“Concentrated” solutions: add more solute or reduce solvent
“Saturated” solutions: no further solute will dissolve in solvent
Exception to this = Supersaturation (but we’ll leave this for another discussion)
Photo Credit:

6. Concentration Concentration may be reported in multiple ways (units):
Molarity (Moles/Liter)
Normality (moles of active ions/Liter)
Percent composition (%); or Parts per…(ppt, ppm, ppb)
Mass per unit volume (grams/L; mg/mL)
Concentration measurements can be reported in several ways; some of these include: (see screen)
In this class, you will use percent composition & mass per unit volume most often
These two measurements tend to be used for more complex solutions (i.e., solutions with molarities that are not easily determined)

7. Dilution
Dilution: using the solvent to increase the volume of the solution; this decreases the solute concentration in the solution
Why would I ever need to make a dilution?
Simple Dilution: dilution of a single solution to make a diluted solution
Serial Dilution: series of dilutions resulting in progressively diluted versions of the Primary (“original” or “stock”) solution.
One of the benefits of working with liquid solutions is the ability to dilute them.
Dilution is a technique that uses a solvent to increase the volume of a solution and thus decrease the concentration of that solution.
It is a concept used in everyday life as well:
If your coffee is too strong, you add water to dilute it and make it more palatable. Many people do this with their juice or other beverages. It can also happen inadvertently when your ice melts and makes your favorite carbonated soft drink taste less sugary.
Why would I ever need to make a dilution?
Some solutions call for solute amounts too small to weigh out (e.g., μg or ng): In science diluting solutions has practical applications as well. Often times you will need a small volume of a low concentration solution. When you do the math you find that you need to weigh out microgram or nanogram amounts of the compound. Lab balances are not up to the task.
Concentrated stock (“Primary”) solutions are useful for dilutions to desired solution: So, you make a higher concentration solution, and then dilute it to the concentration you need.
The more concentrated solution is called a stock (or Primary) solution. You can then dilute the stock solution to the concentration you need, which is often referred to as a working concentration or final concentration.
Different types of Dilutions: Simple Dilution; Serial Dilution… (see screen)

8. Simple Dilution
Simple Dilution: dilution of a single solution to make a diluted solution
1 can concentrated juice (“stock solution”)
4 cans water
1:5 or 1/5th simple dilution factor
Example of Simple Dilution: Concentrated orange juice
Dilution Factor:
Dilution Factor: (a.k.a. dilution ratio)
(Volume of stock solution)
(Volume of stock solution) + (Volume of solvent)
Photo Credit:

9. Serial Dilution
Serial Dilution: series of dilutions resulting in progressively diluted versions of the Primary (“original” or “stock”) solution.
Typically made in increments of 1000, 100, or 10 (logarithmic scale)
Now we face a problem. There are very few scales that can measure that small of a
mass of a compound. And while some micropippettes can accurately transfer one
microliter, it is such a small volume that there is a high rate of error. So, what do we do?
We perform serial dilutions.
common problem faced in a biomedical research
lab. While using very little of a compound is always a financial benefit, it can be a
practical hurdle. When you need a volume or an amount that is too small to accurately
measure, you have a couple of options. You can make a much larger volume of the
solution that you need at the proper concentration. For example we needed 1.5
micrograms of NaCl for a 1ml solution. We could make ten liters of the solution and
weigh out grams of NaCl . But, then will have nine point nine nine nine liters of
our solution left over. A ten liter carboy is quite the space eater in a lab!
The other option is to make a small volume of a much higher concentration solution.
Then you can dilute that solution down to the concentration that you need. Often times
you will need to dilute the higher concentration solution a significant amount which
would again require making a larger volume of the solution at the concentration you
need. However, we can use a series of smaller dilutions into smaller volumes to get to
the volume and concentration that we desire. This is known as using serial dilutions.
While serial dilutions are a great way to save on both reagents and space in the lab,
they have other experimental uses as well. We won’t be covering those uses in this
Webinar, but it is likely you will come across their uses if you work in the lab long
enough.
1:10
1/10th
0.1
101
1:100
1/100th
0.01
102
1:1000
1/1000th
0.001
103
1:10000
1/10000th
0.0001
104
Dilution Factors:

10. Spectrophotometry Check your dilution concentrations…
As a check on your technique, you will use spectrophotometric techniques to measure the strength of your dilutions.
Check your dilution concentrations…
Photo Credit: chemwiki.ucdavis.edu

11. Spectrophotometry
Spectrophotometry: method employing the use of a spectrophotometer to measure the quantity of light that is absorbed by a chemical
Absorbance
Transmittance
This is accomplished by passing a beam of light (at a known wavelength) through a sample and measuring the intensity of light that reaches the detector on the other side of the sample
The beam of light consists of a stream of photons. When a photon encounters an analyte molecule (the analyte is the molecule being studied), there is a chance the analyte will absorb the photon. This absorption reduces the number of photons in the beam of light, thereby reducing the intensity of the light beam that is transmitted.
Every chemical compound absorbs, transmits, or reflects light (electromagnetic radiation) over a certain range of wavelength. Spectrophotometry is a measurement of how much a chemical substance absorbs or transmits. Spectrophotometry is widely used for quantitative analysis in various areas (e.g., chemistry, physics, biology, biochemistry, material and chemical engineering, clinical applications, industrial applications, etc). Any application that deals with chemical substances or materials can use this technique. In biochemistry, for example, it is used to determine enzyme-catalyzed reactions. In clinical applications, it is used to examine blood or tissues for clinical diagnosis. There are also several variations of the spectrophotometry such as atomic absorption spectrophotometry and atomic emission spectrophotometry.

12. Light Waves
Electromagnetic Spectrum:
390nm to 780nm

13. When Light Strikes Matter…
Transmitted
Reflected
Be transmitted through the solution
Be reflected by the solution
Be scattered & attenuated by the solution
Be absorbed by the solution
Refracted
Absorbed

14. Polychromatic Light
Polychromatic Light: light that exhibits more than one color (more than one wavelength)
“White Light” is the most inclusive polychromatic light (being made of all of the visible light spectrum)

15. Polychromatic Light Absorption of specific wavelengths:
The red end of the spectrum has been absorbed
Blue light has been transmitted

16. Polychromatic Light

17. Monochromatic Light Photo Credit: chemwiki.ucdavis.edu
In spectrophotometry, we can gain greater sensitivity by directing a single wavelength through the solution (for this lab, red light through the solution because CuSO4 absorbs strongest at the red end of the visible spectrum).
But to do this, we have to isolate the red wavelengths!
In a spectrophotometer, a light source gives off white light which strikes a prism, separating the light into it component wavelengths.
I0 is the incident light and represents 100% of the light striking the sample. I is the transmitted light. This is the light which has not been absorbed by the sample and will strike the photocell. The photons of light which do strike the photocell will be converted to electrical energy. This current which has been produced is very small and must be amplified before it can be efficiently detected by the galvanometer and displayed for you to read.
Photo Credit: chemwiki.ucdavis.edu

18. Cuvettes & the Blank Blank Solution Cuvette
In order to effectively use a spec. we must first zero the machine. We do this using the blank. The blank contains everything except the compound of interest which absorbs light. Thus, by zeroing the machine using the blank, any measured absorbance is due to the presence of the solute of interest.
Solution
Cuvette
Photo Credit: faculty.Virginia.edu/analyticalchemistry

19. But what does any of this have to do with Solutions & Dilutions?
Beer’s Law
Absorbance is directly proportional to the concentration of a solution.
“The thicker the glass; the darker the brew,
The less the light that passes through!”
Beer’s Law (a.k.a. Beer-Lambert Law)
From:
A spectrophotometer is an instrument that measures the intensity of the light entering a sample and the light exiting a sample and compares the two intensities. Information about the two intensities can be expressed as transmittance (the ratio of the intensity of the exiting light to the entering light) or percent transmittance (%T). Different materials absorb different wavelengths of light. Therefore, the wavelength of maximum absorption by a material is one of the characteristic properties of that material. The %T can be related to the absorbance (A) by the equation below.
A = [log (%T)]
If T = 85%, then
Absorbance = 2 - log[85] = 0.071
Beer's law states that the absorbance is directly proportional to the concentration of a solution. If you plot absorbance versus concentration, the resulting graph yields a straight line. The equation for the straight line (termed regression line) can be used to determine the concentration of an unknown solution once the %T has measured.
The thicker the glass, the darker the brew
The less the light that passes thru…

20. Standardization Graph
In other words… if you plot ABSORBANCE vs CONCENTRATION, the resulting graph yields a straight line.

21. Standardization Graph
In other words… if you plot ABSORBANCE vs CONCENTRATION, the resulting graph yields a straight line.