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OBJECTIVES:

To learn how to operate spectrophotometer To learn how to measure concentration and absorbance of crystal violet solution by using spectrophotometer. a. To learn how to operate spectrophotometer. b. To learn how to measure concentration of crystal violet solution by using spectrophotometer. c. To know and further understanding the relationship between concentration and absorbance.

TABLE OF REAGENT: LIST OF UNKNOWN. Compound Benzoic acid Phenol 2-Nitrophenol max 230 210.5 272

4-Nitrophenol Aniline Methylene blue,chloridetrihydrate Crystal violet

228 230 661 590

Apparatus required:
Spectrophotometer, quartz crystal sample cells. BACKGROUND

A spectrophotometer is primarily used to identify substances and determine their concentration. "Spectro" refers to the "spectrum" of wavelengths that make up both visible light and electromagnetic radiation above or below this range. The visible range is 0.0004mm (= 400 nanometers = 4,000 Angstroms) - violet - to 0.00076mm (= 760 nanometers = 7,600 Angstroms) - red. Immediately above this range is the "infrared" region of longer

wavelengths, while below it is the shorter wavelength "ultraviolet" region. Ordinary light, such as light from the sun, consists of a mixture of wavelengths that is perceived by the human eye and described as "white light". However, when the component wavelengths are spread out in increasing order, the resulting "spectrum" is perceived as an orderly succession of red, orange, yellow, green, blue, indigo, and violet.

The "photo" in "spectrophotometer" refers to light (or to "photons" of light energy), and "meter" means a device capable of quantitative measurement. Putting that all together, a "spectrophotometer" is a device capable of making quantitative measurements of light intensity at selected wavelengths. Its components include a light source or sources, devices (sometimes incorporating filters) for generating beams of light of specific wavelengths from that source, special holding devices for tubes or "cuvettes" containing samples through which light beams are passed, and detectors that can measure the intensity of light that has passed through a sample.

Some spectrophotometers used in bioresearch are designed for analysis of both ultraviolet (UV) and visible (VIS) regions of the spectrum, while others are VIS range only. The former are more useful because many biomolecules of interest, such as nucleic acids and proteins, absorb wavelengths in the UV range. They are correspondingly more expensive instruments, however. You will be using a Tech Facility VIS-only spectrophotometer. However, if you use a spectrophotometer in a research laboratory, it is likely that it will be UV-VIS.

Identification of compounds: When matter interacts with energy, energy is frequently absorbed. Different types of chemical compounds have distinctive atomic and molecular structure, and will absorb energy at characteristic energy levels and wavelengths. The absorbed energy can result in changes in the energy level of electrons in the atoms comprising a molecule, as well as in vibration and/or rotation of those atoms. In some cases, absorbed light energy is re-emitted as light of lower energy and longer wavelength. This phenomenon is known as "fluorescence". An unknown substance can often be identified by comparing the amount of light of different wavelengths that it absorbs, i.e., its absorption spectrum, with the absorption spectra of known substances. Such an analysis is performed using a spectrophotometer to measure the amount of light absorbed over a range of wavelengths of incident light.

A solution looks colored to the human eye because molecules are absorbing wavelengths corresponding to the other colors. It appears as a particular color because it is NOT absorbing the wavelengths of that color; rather, they are transmitted to your eye. For example, a compound appearing blue in solution is transmitting a range of 435-480 nm wavelengths to the eye, and is typically absorbing at 580-595 (yellow). A compound appearing red is typically transmitting in a range of 610-750 nm, and absorbing in the 595-610 nm range (bluish-green).

Concentration determination: The concentration of an unknown sample can also be determined using a spectrophotometer. When a light beam is passed through a sample, part of it is absorbed, and part is transmitted through the sample. To quantify this effect, the transmittance (T) is defined as the intensity of the transmitted light (It) divided by the original (or incident) intensity of the light beam (Io). In abbreviated notation, T = It/Io. The absorbance (A) is defined as the log10 of the reciprocal of the transmittance, i.e., A = log (1/T). This is a measure of how much light is absorbed by the sample, rather than transmitted through it. Knowing one (A or T) it is easy to calculate the other.

There is a relationship between concentration and absorbance; the higher the concentration of a substance in a solution, the greater the amount of light it will absorb, assuming that the wavelength is an absorbable one for the substance. You will determine the relationship between concentration and absorbance in Exercise #5, below. Since a spectrophotometer measures absorbance (or transmittance, from which absorbance can be calculated), the spectrophotometer can be used to determine an unknown concentration of a substance by comparison with a "Standard Curve", i.e., a graph showing the absorbance produced by various known concentrations of that same substance.

Theory:
A compound absorbs energy in accord with its ability to have an electron excited by a particular wavelength. A plot of wavelength, frequency, or related units versus percent transmission or absorbance will give a curve. Percent transmission and absorbance (A) is defined mathematically as:-

%T = radiation intensity transmitted or reflected (100) / incident radiation intensity

A = log (incident radiation intensity / radiation intensity transmitted or reflected) Absorption of photons is proportional to concentration of sample and path length: A lc A is the absorbance, l is the path length, and c is the concentration. The introduction of constant, epsilon (), changes the proportionality into an equation known as Beers law: A= lc Epsilon is called the molar absorptivity or molar extinction coefficient and its related to the probability that light of given wavelength incident on sample will actually be absorbed by electrons in molecule. Absorptivity is property of given compound but depends on wavelength and solvent. The solvent for spectroscopy should be transparent (nonabsorbent) over the entire spectral region to examine. Because they possess only sigma bonds which do not absorb in the visible and near UV ranges. Also, the solvent should be very pure, because some impurities absorb very intensely. So it would alter the appearance of, or even obscure the spectrum of the compound of interest.

RESULT AND CALCULATION:

Sample Blank 1 2 3

Concentration 0 0.001 0.007 0.01

Absorbance 0 0.076 0.137 0.216

Sample Blank Unknown 0.008 0.08

Absorbance 0 0.025 0.045 0.366

Concentration 0 0.001 0.017 0.002

3. RESULT
a. Measuring concentration. Num 1 2 3 Calculation: 1. Concentration x General equation ; y = 2.410137*x Absorbance 0.025 0.045 0.365 Concentration x x=0.025/2.410137 =0.010 x=0.045/2.410137 =0.019 x=0.365/2.410137 =0.151 . % Error |0.001-0.010|x100=9% 0.001 |0.007-0.019|x100=1.71% 0.007 |0.010-0.151|x100=14.1% 0.010 Cell 1 2 3 Concentration 0.001 0.007 0.010 Absorbance 0.075 0.139 0.214

3. If epsilon is 20 000 and a 1.00cm cell is being used, what molar concentration would be necessary to obtain an absorbance of 1.00? A = lc C = A/l = 1.00/(20000*0.01m) = 0.005

Observation:
When the first step was performed, i.e. when the cell containing the deionized water was placed in the cell holder, the computer performed some calculations regarding some corrections involving the reference sample. After doing that, the computer asked for another sample of the blank cell which was followed by a sample of the methylene blue. The computer then calculated the wavelength of the light absorbed by the cell and its constituent and calculated the absorbance. This was followed by another sample solution, and the computer performed the reading for that as well. As only two solutions were tested, besides the ones containing deionized water, we proceeded onto the next step of the analysis involving the concentration of the sample. Again the same observations were made and at the end of the process the computer plotted a graph between the concentration of the sample and the wavelength.

DISCUSSION:
Since this experiment involved high level gadgetry, most of the work was performed by the computer. None the less, the whole experiment came as a learning experience as the techniques, procedure and precautions involved while dealing with a spectrophotometer were carefully noted down and understood. Furthermore the importance of the utility of spectroscopy was realized as spectroscopy is a very professional and precise way to identify unknown compound and to calculate their concentration based on their phenomena of absorbing particular wavelengths of light. As the wavelength absorbed is attributing to that particular solution, it can be simply identified and the required calculations can be made. The spectrophotometer is based on a very interesting principle. It has a light chamber in its housing which emits light rays of all wavelengths in the spectrum and calculates which wavelength was absorbed, and based on that calculates the concentration and nature of the sample solution. The problems faced, was only in using the spectrophotometer. Regarding the precautions to be kept in mind, there are quite a few, First of all are not to hold the sample cells on the clear sides of it. These two sides are going to be ones where the beam is going to pass through. Touching them will cause our fingerprints printed on it. Consequently, the glass will not be clean and some particles of the light beam are going to be reflected by the fingerprints. Usage

the spectrophotometer also requires full care. The beam chambers should not be played with and the cell holder should be taken care not to break it while placing the sample cells. Even the cells are easily broken. So we cannot hold it so hard that it might break. There must also be no spills in the chamber. This requires that we should not fill the cell with our sample solution more than of the cell height and it should be closed. This is because of the possibility that the beam may cause the solution to rise up. But we should also not be so stingy filling up our cell because too less might cause the beam to pass through nothing but air. At the end of the sequence, the computer plotted a graph between the wavelength and the concentration which turned out to be linear. However, as the experiment was performed only to identify the concentration of the crystal violet, which was supposed to be the unknown but was already disclosed to us, we only found its relation with the wavelength. Also, we were able to calculate Epsilon using Beers low for the unknown sample and the standards.

DISCUSSION:
A spectrophotometer is primarily used to identify substances and determine their concentration. "Spectro" refers to the "spectrum" of wavelengths that make up both visible light and electromagnetic radiation above or below this range. The visible range is 0.0004mm (= 400 nanometers = 4,000 Angstroms) - violet - to 0.00076mm (= 760 nanometers = 7,600 Angstroms) - red. Immediately above this range is the "infrared" region of longer wavelengths, while below it is the shorter wavelength "ultraviolet" region. Ordinary light, such as light from the sun, consists of a mixture of wavelengths that is perceived by the human eye and described as "white light". However, when the component wavelengths are spread out in increasing order, the resulting "spectrum" is perceived as an orderly succession of red, orange, yellow, green, blue, indigo, and violet.

The "photo" in "spectrophotometer" refers to light (or to "photons" of light energy), and "meter" means a device capable of quantitative measurement. Putting that all together, a "spectrophotometer" is a device capable of making quantitative measurements of light intensity at selected wavelengths. Its components include a light source or sources, devices (sometimes incorporating filters) for generating beams of light of specific wavelengths from that source, special holding devices for tubes or "cuvettes" containing samples through which light beams are passed, and detectors that can measure the intensity of light that has passed

through a sample.

Some spectrophotometers used in bioresearch are designed for analysis of both ultraviolet (UV) and visible (VIS) regions of the spectrum, while others are VIS range only. The former are more useful because many biomolecules of interest, such as nucleic acids and proteins, absorb wavelengths in the UV range. However, in our laboratory, it is likely that it will be UV-VIS. This kind of spectrophotometer is an instrument that emits an electromagnet ray of continually changing wavelength, beginning at one of the spectrum and progressing to the other end. The beam passes through the sample and the instrument measures the amount of radiation that is absorbed. Most modern spectrophotometers have two chambers through which beams are passed : one for sample and one for the pure solvent in which the sample is dissolved. When both chambers are used, the instrument is said to be in double-beam mode. The double-beam mode resuts in automatic subtraction of absorptions due to solvent.

Basically, a solvent for spectroscopy should be transparent (nonabsorbent) over the entire spectral region to be examined. A number of such solvents exist because they possess only single bond which is sigma bonds, which do not adsorb in the visible and near UV ranges. Good solvents include cyclohexane, ethanol, methanol and water. We should use solvents that are of spectral grade or very pure, and handle them so they do not become contaminated. Because some impurities absorb very intensely, tiny amounts of such an impurity introduced into the solvent along with the sample could produce a spectrum that would alter the appearance of, or even obscure ,the spectrum of the compound of interest.

There is a relationship between concentration and absorbance; the higher the concentration of a substance in a solution, the greater the amount of light it will absorb, assuming that the wavelength is an absorbable one for the substance. Absorption of photons is proportional to concentration of the sample and to path length: A lc A is the absorbance, l is the path length in cm and c is the concentration in moles per liter. The introduction of a constant epsilon (c), changes the proportionality into an equation known as Beers law A= lc Epsilon has units o 1/cm x mole because absorbance is unit less, being a logarithmic function of the ratio of incident to transmitted light. Epsilon is called the molar absorptivity or molar

extinction coefficient and is related to the probability that light of iven wavelength incident on the sample will actually be absorbed by the electrons in the molecule. Absoptvity is a property of a given compound but depends on wavelength and solvent. Ordinarily absorptivity is reported for each maximum in the curve and can be determined by measuring the absorption for a known concentration over a known path length. Rearranged the equation will gives: = A/lc when c is measured at max, it is often referred to as max. Once epsilon is known for a given compound,it can be used to determine concentrations.

In our experiment, the result that we get for the concentration and absorption of unknown sample was not really accurate. Not all the points that we plot can gives a linear curve. Only a few points is correct value This is happen because we are conducted it wrongly. On the other hand, the spectrophotometer is an effective device to measure the concentration and absorption. In order to get the correct result, there are some precaution that need to be considered. First, make sure to close the spectrophotometer before start any process. Second, wipe out the clear suface of the cuvette with tissue paper before you put it in the cuvette holder. If the surface had been contaminated, it cannot transmit the light properly. So,it will affected our result. Never wipe using something that can scratch the clear surface. Lastly, make sure to put the the sample solution of the cuvette. It will spilled out from the cuvette if we put it exceed than . So, we need to know the technique to used the pipette in order to transfer the sample into the cuvette.

4. DISCUSSION
A spectrophotometer is an instrument designed to detect the amount of radiant light energy absorbed by molecules. When light is reflected from a diffraction grating, it is split into its component colors or wavelengths, which then diverge. Sections of the projected spectrum can be either blocked or allowed to pass through the slit so that only one wavelength will pass to the other sections of the spectrophotometer. Light that passes through the slit travels to the phototube, where it creates an electric current proportional to the number of photons striking the phototube. If a digital meter is attached to the phototube, the electric current output can be measured and recorded. The scale is usually calibrated in two ways: percent transmittance, which runs on a scale from 0 to 100; and absorbance, or optical density units, which runs from 0 to 2.

A compound absorbs energy in accords with its ability to have an electron excited by a particular wavelength. A plot of wavelength, frequency, or related units versus percent transmission or absorbance will give a curve. A=log (incident radiation intensity/ Radiation intensity transmitted or reflected) An electronic spectrum of a compound is not a single, sharp peak but is more or less smooth curve representing absorptions over a rather wide range of wavelength. The reason for this wide range is that atoms of molecules are arranged in an almost infinite number of positions with respect to each other because of vibrations of the atoms as their bonds stretch, bend and twist. The electrons between atoms that are stretched apart require excitation by a different quantum of energy than electrons between atoms with a more contracted bond. Electrons that are twisted or bent are excited by photons that are different from those in straighter bonds. Therefore, the spectrum is a composite of thousands of absorptions over a range of wavelengths. Absorption of photons is proportional to concentration of the sample and to path length. A lc In choosing a solvent, it should be transparent over the entire spectral region to be examined. A number of such solvents exist because they posses only sigma bonds, which do not absorb in the visible and near UV ranges. All of the weakness while doing the experiment has been analyzed and action to improve has been recognized for better future experiment. First and foremost, I should fully understand what is the objective of the experiment and manual should be read before doing the experiment. Any confusion should be asked to the demonstrator or lectures so that the flowing of the experiment run out smoothly. Because not fully understand, I have made wrong interpretation regarding the relationship between the concentration and absorbance. Some of the strictly precaution that are being highlighted in the manual is that the method on how you are holding the cuvette.The cuvette should be hold and the line part not the tranparence part.By holding at the line part the sample can be clearly seen through the non line part. Besides that, the amount of diluted crystal violet that can be inserted is of the cuvette. So, it will not spill. Another precaution is that you should wipe the excess outside the cuvette by using tissue to avoid any injury to our skin if the chemical compound is strong acid. In measuring the concentration by using spectrophotometer, the cells must be clean.Rinse them several times with solvent before use, between successive determinations and after use. When filling a cell, you should not let solution run down the outside of the clean cell.

CONCLUSION:
As conclusion, we had achieved the objectives of the experiment. We now understand the using of spectrophotometer and how to handle it. We also understand how to calculate the

concentration and absorbance of unknown compound using this device. Finally, this experiment was done successfully.

5. CONCLUSION.
After completing the experiment, my understanding on how the spectrophotometer is functioning is increases by the clear and complete explanation by the lecturer and demonstrator. In addition, I also can handle the spectrophotometer in case to measure concentration of other chemical compound in another future experiment. The most important thing when you are dealing with chemical compound is you should be calm and concentrate in what you are doing either you are inserting the chemical from beaker to cuvette or you want to insert the value into the spectrophotometer. This is the good ethics to avoid any accident in the laboratory. Finally, I can conclude that concentration is directly proportional to absorbance. The absorbance will increase if the concentration is high. The plotted graph is a linear graph. This is the same as relationship between the absorbance and transmission. Higher the absorbance, transmission will be high as well. If the absorbance is low, transmission also are less.

6. QUESTIONS.
1. Why must a solvent used for spectrophotometer be pure? A solvent should be pure to avoid it from being contiminated.This is because some impurities absorb very intensely, tiny amounts of such an impurity introduced into the solvent along with the sample could produce a spectrum that would alter the appearance of the spectrum of the compound of interest.

2. What is the spectral region for interpreting visible spectra? The spectral region for interpreting visible spectra is the central region where the wavelength is the highest. 3. What parts of sample cells should not be touched? The clear part of the cuvette should not be touched. 4. What precaution must be taken when inserting cells into the cell compartment? When inserting cells into the cell compartment, do not let solution down the outside of the clean cell. The clear part should be put at the right part in the cell compartment, so that light can passes through.

QUESTIONS:
1) Why must a solvent used for spectrophotometer be pure? Because impurities in solvent can absorb very intensely and could produce a spectrum that would alter the appearence of the spectrum of compound interest. 2) What is the spectral region for interpreting visible spectra?

The spectral region for interpreting visible spectra is the central region where the wavelength is the highest. 3) What parts of sample cells should not be touched? The parts of sample cells should not be touched is the clear sides of the cells. 4) What precaution must be taken when inserting cells into the cell compartment? The precaution that must be taken when inserting cells into the cell compartment is we must make sure that there is no spilt solution or any solution run down at the outside of the clean cell.

5) Calculate the concentration (x) by using the equation: y = 2.410137*x, where y is absorbance. Sample 1 2 3 4 5 6 Absorbance, y 0.076 0.137 0.216 0.025 0.045 0.366 Calculation, x = y / 2.410137 = 0.076 / 2.410137 = 0.137 / 2.410137 = 0.216 / 2.410137 = 0.025 / 2.410137 = 0.045 / 2.410137 = 0.366 / 2.410137 Concentration, x (mg/mL) 0.032 0.057 0.089 0.010 0.019 0.152

6) Calculate the % error for the result of your esperiment

Sample

1 2 3 4 5

Concentration (theory) (mg/ml) 0.001 0.007 0.010 0.0012 0.0021

Concentration (experimental) (mg/ml) 0.032 0.057 0.089 0.010 0.019

Calculation [(0.001- 0.032)] x 100 0.001 [(0.007 - 0.057)] x 100 0.007 [(0.010 - 0.089)] x 100 0.010 [(0.0012- 0.010)] x 100 0.0012 [(0.0021- 0.019)] x 100

3100 714 790 733 1899

0.0021 6 0.0154 0.152 [(0.0154 - 0.152)] x 100 0.0154 887

7) If epsilon is 20000 and a 1.00 cm cell is being used, what molar concentration would be necessary to obtain an absorbance of 1.00? A = lc c = A / l = (1.00) / (20 000 x 1.00cm) c = 5 x 10-5 M

EXERCISE
Q. Why must a solvent used for spectrophotometer be pure? Ans. A solvent used for spectrophotometer should be pure because some impurities absorb very intensely and tiny amounts of such impurities introduced into the solvent along with the sample could produce a spectrum that would alter the appearance of the compound of interest. Q. What is the spectral region for interpreting visible spectra? Ans. Zero Q. What parts of sample cells should not be touched? Ans. The clear part of the sample cells should not be touched as it leaves fingerprints on the crystal made sample cells. Only the translucent sides (i.e. the smoked sides) should be used to handle the sample cells. Q. What precaution must be taken when inserting cells into the cell compartment? Ans. 1. The cells should be held only along the smoked side and not the clear side. 2. It should be made sure that the solution is pure. 3. The sample solution should not be spilled on the quartz cell as that could lead to erroneous readings. 4. Make sure that the clear side of the cells face the light chamber and not the smoked sides. 5. Fill only three quarters of the quartz cells with the sample solution. 6. Handle the cells carefully because they are expensive.

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