Uv vis spectroscopy practical

[SALUM MKATA B.PHARM 3]

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DATE: May 28, 2014.

ULTRA-VIOLET VISIBLE SPECTROSCOPY PRACTICAL REPORT:

Aim: To apply the Beer-Lambert relationship to an aqueous solution containing

an absorbing substance and thus determine its respective concentrations.

INTRODUCTION AND THEORY:

Ultraviolet–visible spectroscopy or ultraviolet-visible spectrophotometry (UV-

Vis or UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in the

ultraviolet-visible spectral region (about 190-820nm).The Ultraviolet-visible

absorption based on molecules containing π-electrons or non-bonding electrons (n-

electrons) which can absorb the energy in the form of ultraviolet or visible light to

excite these electrons to higher anti-bonding molecular orbitals. The more easily

excited the electrons (i.e. lower energy gap between the HOMO and the LUMO), the

longer the wavelength of light it can absorb.

UV/Vis spectroscopy is routinely used in analytical chemistry for the quantitative

and qualitative determination of different analytes, such as transition metal ions,

highly conjugated organic compounds, and biological macromolecules.

Spectroscopic analysis is commonly carried out in solutions but solids and gases may

also be studied

The ultraviolet region (about 400-190) is particularly important for the qualitative

and quantitative determination of many Organic compounds, especially those with a

high degree of conjugation, also absorb light in the UV or visible regions of the

electromagnetic spectrum. The solvents for these determinations are often water for

water-soluble compounds, or ethanol for organic-soluble compounds. While in the

visible region (about 400-820nm), spectrophotometry methods are widely used for

the quantitative determination of many trace substances, especially inorganic species.

The basic principle of quantitative absorption spectroscopy lies in comparing the

extent of absorption of a sample solution with that of a set of standards under

radiation of a selected wavelength through the application of Beer-Lambert law.

The Beer-Lambert law states that the absorbance of a solution is directly

proportional to the concentration of the absorbing species in the solution and the path

length. Thus, for a fixed path length, UV/Vis spectroscopy can be used to determine


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the concentration of the absorber in a solution. It is necessary to know how quickly

the absorbance changes with concentration.

Equation:

A =

Where:

A= absorbance

P= power of transmitted

PO =power of incident

ϵ= extinction coefficient (mol-1

dm3cm

-1)

b= path length of cell (cm)

c= concentration of absorbing species.

Note: In many cases, the sample compound does not absorb radiation appreciably in

the wavelength provided, it’s then necessary to form an absorbing substance by

reacting a compound in question with other reagents.

What a special about Ultraviolet-visible spectrometer:

The instrument used in ultraviolet-visible spectroscopy is called a UV/Vis

spectrophotometer. It measures the intensity of light passing through a sample ( ),

and compares it to the intensity of light before it passes through the sample ( ). The

ratio is called the transmittance, and is usually expressed as a percentage (%T).

The absorbance, , is based on the transmittance:

The UV-visible spectrophotometer can also be configured to measure reflectance. In

this case, the spectrophotometer measures the intensity of light reflected from a

sample ( ), and compares it to the intensity of light reflected from a reference


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material ( ) (such as a white tile). The ratio is called the reflectance, and is

usually expressed as a percentage (%R).

The basic parts of a spectrophotometer are; a light source, a holder for the sample,

a diffraction grating in a monochromator or a prism to separate the different

wavelengths of light, and a detector. The radiation source is often a Tungsten

filament (300-2500 nm), a deuterium arc lamp, which is continuous over the

ultraviolet region (190-400 nm), Xenon arc lamp, which is continuous from 160-

2,000 nm; or more recently, light emitting diodes (LED) for the visible wavelengths.

The detector is typically a photomultiplier tube, a photodiode, a photodiode array or a

charge-coupled device (CCD).

Single photodiode detectors and photomultiplier tubes are used with scanning

monochromators, which filter the light so that only light of a single wavelength

reaches the detector at one time. The scanning monochromator moves the diffraction

grating to "step-through" each wavelength so that its intensity may be measured as a

function of wavelength. Fixed monochromators are used with CCDs and photodiode

arrays. As both of these devices consist of many detectors grouped into one or two

dimensional arrays, they are able to collect light of different wavelengths on different

pixels or groups of pixels simultaneously.

Figure.1


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APPARATUS AND CHEMICAL USED

Apparatus:

1) Spectrophotometer

2) Cuvettes

3) Measuring flask (10mls.)

4)1mls and 10mls capacity pipettes

5) Pipette filler

6) Storage bottle Figure.2 spectrophotometer.

7) Digital mass balance.

8) Test tube rack

9) Beaker.

10) Stopper

11) Stop watch

Figure.3 Digital mass balance

Chemical/ Reagents:

1) Salicylic acid (0.1%)

2) Acetate buffer (0.05M)

3) Distil water


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PROCEDURES:

1) We turn on the spectrophotometer and allow it to warm up for at least 20 min.

Then we determine the absorption spectrum using the standard acetate buffer.

2) We select one of the cuvettes for the blank solution (in this case acetate buffer)

and we do not interchange it with the other cuvettes also we did not handle the

lower portion of cuvettes through which the light passes.

3) We always rinse the cuvettes with several portions of solution by using acetate

buffer before taking a measurement.

4) Then we wipe the outside of cuvettes with tissue paper.

5) Then we inserted the cuvette into the cell holder with the index line facing us

to avoiding scratching.

6) Then we turn the wavelength control knob to 265nm with blank solution

calibrate the spectrophotometer.

A. Preparation of the solution:

1) We prepared a stock solution of 0.1% salicylic acid by: accurately measuring

100mg of salicylic acid by digital mass balance then we dissolved the obtained

mass in 100ml of acetate buffer in beaker.

2) From that stock solution above we prepared serial dilution containing 0.05%,

0.025%, 0.01%, 0.005%, 0.0025, and 0.001%.(Note all percentages are in

weight by volume ).

We apply dilution law of same substance as:

C1 V1=C2V2

Where:

M1=concentration of concentrated (stock) solution.

V1= volume of concentrated (stock) solution.


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M2= concentration of diluted solution.

V2= volume of diluted solution.

Note: V2 will be equal to 10ml because we have to diluting the stock solution by

adding sufficient amount of acetate buffer to make a solution of 10ml in measuring

flask. Also C1 and C2 known then V1 obtained by following formula

3) Then we labeled test tube from 1 up to 6. After obtained required volume of

stock solution to be taken from beaker containing stock solution of salicylic

acid. Then we add sufficient amount of acetate buffer to make 10ml solution

results summarized in following table.

TABLE.1;

S/N 1 2 3 4 5 6

Concentration

(%w/v)

0.05 0.025 0.01 0.005 0.0025 0.001

Volume of

stock solution

need(ml)

5.00 2.50 1.00 0.50 0.25 0.10

Volume of

diluted

solution(ml)

10 10 10 10 10 10

4) Then we prepared (standard blank) in which 20ml of acetate buffer is

substituted for the sample.

B. Determination of absorbance

1) We set blank solution in the cuvette and calibrate at wavelength at wavelength

control of 296 nm.

2) Then we inserted cuvette containing the sample- then we read and recorded the

absorbance for all solutions in above section A.

3) Then we determine the absorbance of unknown sample.


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Results obtained as follows

NOTE: since we have to represents concentration in MOLARITY then:

Concentration in Molarity= Concentration in g/dm3

Molar

mass in g/mol

Concentration in g/dm3:

Since

Y g in 1000 cm3

X g in 100 cm3

Since X is mass of sample in above table then mass Y which is in 1000cm3

can be obtain.

But molar mass of salicylic acid= 138g/mol.

Then we change those concentrations in w/v% in Table 1 above into Molarity

Then we obtain Table below.

TABLE .2;

TRIAL CONCENTRATION(MOL/LITRE) ABSORBANCE

1 7.24 x 10-5

0.007

2 1.81x 10-4

0.003

3 3.62x 10-4

0.007

4 7.24x10-4

0.011

5 1.81x10-3

0.015

6 3.62x10-3

0.018

Unknown Z 0.042


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DISCUSSION:

We plot the graph of Absorbance vs. concentration in Excel sheet as shown below:

ANSWERS TO GIVEN QUESTIONS AND SOURCES OF ERROR:

1) Yes, it can, if an appropriate mathematical model can be fitted to the data

obtained from standards.

For example the older versions of the Sedex Evaporative Light-Scattering

Detector (ELSD) (image below) gave quadratic response to most analytes, so

one had to use a quadratic fit for the standards, and calculating the

concentration of unknowns required solution of by the quadratic formula.

Figure.4

y = 3.7129x + 0.006 R² = 0.8294

0

0.005

0.01

0.015

0.02

0.025

0 0.0005 0.001 0.0015 0.002 0.0025 0.003 0.0035 0.004

Ab

sorb

ance

concentration (mol/litre)

ABSORBANCE VS. CONCENTRATION (MOLE/LITRE)


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In NIR spectroscopy, non-linear fit methods such as Principal Component

Analysis and Multiple Linear Regression are often used because the sample

response may not strictly obey Beer's law, especially for reflectance spectra.

2) There no any added advantage of using choosing a wavelength at

absorbance plateau other than the maximum since:

It really depends on what is the largest source of error. Taking the readings at

the peak maximum is best at low absorbances because it gives the best signal-

to-noise ratio, which improves the precision of measurement. If the dominant

source of noise is photon noise, the precision of absorbance measurement is

theoretically best when the absorbance is near 1.0. So if the peak absorbance is

below 1.0, then using the peak wavelength is best, but if the peak absorbance is

well above 1.0, you might be better off using another wavelength where the

absorbance is closer to 1. Another issue is calibration curve non-linearity,

which can result in curve-fitting errors.

The non-linearity caused by polychromatic light is minimized if you take

readings at either a peak maximum or a minimum, because the absorbance

change with wavelength is the smallest at those wavelengths. On the other

hand, using the maximum increases the calibration curve non-linearity caused

by stray light.

But, Very high absorbances cause two problems:

The precision of measurement is poor because the transmitted

intensity is so low, and the calibration curve linearity is poor due to

stray light. The effect of stray light can be reduced by taking the

readings at a wavelength where the absorbance is lower or by using

a non-linear calibration curve fitting technique. Finally,

If spectral interferences are a problem, the best measurement

wavelength may be the one that minimizes the relative contribution

of spectral interferences (which may or may not be the peak

maximum). In any case, don't forget: whatever wavelength you use,

you have to use the exact same wavelength for all the standards and

samples.


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3) Cuvettes may be circular, square or rectangular (the latter being uncommon),

and must be constructed of a material that will transmit both the incident and

emitted light. Square cuvettes or cells will be found to be most precise since

the parameters of path length and parallelism are easier to maintain

during manufacture. However, round cuvettes are suitable for many

more routine applications and have the advantage of being less expensive.

Figure 5.Examples of sample cells for UV/Vis spectroscopy. From left to right

(with path lengths in parentheses): rectangular plastic cuvette (10.0 mm),

rectangular quartz cuvette (5.000 mm), rectangular quartz cuvette (1.000 mm),

cylindrical quartz cuvette (10.00 mm), cylindrical quartz cuvette (100.0 mm).

4) The Beer-Lambert Law will not be obeyed if

The photons of light striking the detector do not all have an equal chance

of absorption by the sample. This can happen if they have different

absorption coefficients, different path lengths through the sample, or if

they encounter different concentrations of sample molecules. Also

If anything else is present in the sample that absorbs light or causes

light scattering, the measured absorbance will not be zero when the

analyte's concentration is zero, contrary to Beer's Law.

If the absorber undergoes any type of chemical reaction or equilibrium

that varies as a function of concentration, Beer's Law will not be obeyed

with respect to the overall or total concentration, because the


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concentration of the actual absorbing molecule is not proportional to the

overall concentration of the solution. The "c" in Beer's Law refers to the

concentration of just the absorber, not to the total concentration of all the

compounds reacting with or in equilibrium with the absorber. Even if

Beer's Law holds exactly for each individual compound, the total

absorbance of the mixture will not follow Beer's Law with respect to the

total concentration if the proportion of each compound changes with

concentration (unless by chance the absorptivity of all those compounds

happens to be exactly the same).

Deviations in absorptivity coefficients at high concentrations (> 0.01M)

due to electrostatic interactions. Changes in refractive index at high

analyte concentration.

Fluorescence or phosphorescence of the sample and Non-

monochromatic radiation.

5) From the graph we obtain the equation below:

Y = 3.7129X + 0.006

Hence the unknown concentration can be obtained since:

Y=value from y-axis i.e. the absorbance

X=value from x-axis i.e. the concentration in Mol/litre

But absorbance (Y value) of unknown sample is obtain which is equal to 0.042

Then we can find the value of unknown concentration!

Take

0.042=3.7129X+ 0.006

Then

0.042-0.006=3.7129X

0.036=3.7129X

Then divide by 3.7129 both side to obtain X=9.696x 10-3

There fore

Unknown concentration in mol/litre of solution is equal to 9.696x 10-3


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6) The molar extinction coefficient of SA

From below equation

A

Where:

A= absorbance

ϵ= extinction coefficient (mol-1

dm3cm

-1)

b= path length of cell (cm)

c= concentration of absorbing species.

Take

A1 =0.007 and A2=0.003

Also

C1= 3.62x 10-4

and C2=1.81x 10-4

b =1cm

A1=ϵ1c1b and A2=ϵ2C2b

0.007= ϵ1 x 3.62x 10-4

x1cm …………………………………….eqn.1

0.003= ϵ2x1.81x 10-4

x1cm…………………………………………eqn.2

Then

ϵ1=19.33

ϵ2=16.57

Then molar absorbivity of SA Will be 19.33+16.57=35.9

35.9/2=17.95moll-1

cm-1


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Source of errors:

1) Stray Light: A problem when working at limits of a spectrometer’s range.

2) Cells and Solvents: Everything else except the sample should be as

transparent as possible.

3) Sample Preparation: If two samples are prepared so that one carries along a

greater concentration of insoluble particulates, then additional scattering will

lead to an apparent greater absorption.

4) Slit Width Affects Absorbance Measurements: If a significant variation in

absorptivity occurs over the spectral bandwidth admitted by the slit, a non-

linear variation (non-Beer’s Law) with concentration will be observed. This

arises because the spectrometer measures the average transmissivity over the

spectral bandwidth, but transmissivity and concentration are not linearly

related. Keep slit width large to increase S/N ratio, but must keep it small

enough to maintain a linear relationship with concentration changes. This

effect is minimized if the absorptivity changes slowly with wavelength. Select

a wavelength near a peak maximum. Use a slit width to provide a bandwidth

about 1/10 the spectral feature width.

5) Wavelength error: In liquids, the extinction coefficient usually changes

slowly with wavelength. A peak of the absorbance curve (a wavelength where

the absorbance reaches a maximum) is where the rate of change in absorbance

with wavelength is smallest. Measurements are usually made at a peak to

minimize errors produced by errors in wavelength in the instrument, that is

errors due to having a different extinction coefficient than assumed.

CONCLUSION, ACKNOWLEDGEMENT AND REFERENCES:

CONCLUSION:

From above we can conclude that UV/Vis spectroscopy is best method

which routinely used in analytical chemistry for the quantitative

determination of different analytes, such as transition metal ions, highly

conjugated organic compounds, and biological macromolecules.


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Spectroscopic analysis is commonly carried out in solutions but solids and

gases may also be studied.

A UV/Vis spectrophotometer may be used as a detector for HPLC. The

presence of an analyte gives a response assumed to be proportional to the

concentration. For accurate results, the instrument's response to the analyte

in the unknown should be compared with the response to a standard; this is

very similar to the use of calibration curves. The response (e.g., peak

height) for a particular concentration is known as the response factor.

ACKNWOLEDGEMENT:

1. TO MR. EDSON-LAB. TECHNICIAN

2. TO Dr. E. KAALE.

3. TO Dr. SEMPOMBE-HEAD OF DEPARTMENT OF MEDCINAL CHEMISTRY

4. TO MY FELLOW GROUP MEMBER AND THE REST OF CLASS.

REFERENCES:

1) http://en.wikipedia.org/wiki/Molar_extinction_coefficients

2) http://www.thestudentroom.co.uk/showthread.php?t=1424595

3) https://answers.yahoo.com/question/index?qid=20130721231123AABslPR

4) http://chemwiki.ucdavis.edu/Organic_Chemistry/Organic_Chemistry_Wit

h_a_Biological_Emphasis/Chapter__4%3A_Structure_Determination_I/S

ection_4.3%3A_Ultraviolet_and_visible_spectroscopy

5) http://en.wikipedia.org/wiki/Cuvette

6) Practical protocol by Department of Medicinal chemistry-School of

Pharmacy, MUHAS.

http://en.wikipedia.org/wiki/Molar_extinction_coefficients

http://www.thestudentroom.co.uk/showthread.php?t=1424595

https://answers.yahoo.com/question/index?qid=20130721231123AABslPR

http://chemwiki.ucdavis.edu/Organic_Chemistry/Organic_Chemistry_With_a_Biological_Emphasis/Chapter__4%3A_Structure_Determination_I/Section_4.3%3A_Ultraviolet_and_visible_spectroscopy



http://en.wikipedia.org/wiki/Cuvette

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Uv vis spectroscopy practical