Principles of Spectrophotometer (1)

Title Objective : : Principles of Spectrophotometer (1)

1) To introduce the usage of spectophotometry 2) To determine the extinction coefficient ( E ) of compound given.

Introduction : The use of a spectrophotometer. Spectrophotometer is a photometer (a device for measuring light intensity) that can measure intensity as a function of the color, or more specifically, the wavelength of light. There are many kinds of spectrophotometers. Among the most important distinctions used to classify them are the wavelengths they work with, the measurement techniques they use, how they acquire a spectrum, and the sources of intensity variation they are designed to measure. Other important features of spectrophotometers include the spectral bandwidth and linear range.Perhaps the most common application of spectrophotometers is the measurement of light absorption, but they can be designed to measure diffuse or specular reflectance. Strictly, even the emission half of a luminescence instrument is a kind of spectrophotometer. Meterial : Spectrophotometer,plastic cuvvate,glass cuvvate,distilled water,1.0mg/ml bovine serum ( BSA ), 6,0 mg/l adenine,1 x10-5 cytocrome Method
Method :

1. A cuvette was filled with bovine serum albumin ( BSA ) 2. The spectrophotometer was set at 280 nm and the reading set to zero with distilled water. 3. Then the absorbance of 1.0 mg / ml BSA was measured and the extinction coefficient for BSA was determined by the formula given. 4. The above step was repeated using 6.0 mg / l adenine with absorbance at 261 nm and 1 x 10 M cytochrome with absorbance at 450 nm.

Result :
Solution Bovine serum albumin ( BSA ) Adenine Cytochrome Absorbance 1.177 0.0517 0.640

a) Bovine Serum Albmin ( BSA ) A ( absorption ) : 1.177 MW : 66000 C : 1.0 mg/ml I : 280 nm

1 mg = 0.001 g (1g/1000mg) 1g/100ml = g in 100ml 1mg/ml BSA = 0.001 g/ml Hence,C = 0.1g/1000ml Mass = 1g/1000ml
1%

Formula for percentage :

E
1cm

E = A/C = 1.177/0.1 = 11.77 mg cm-1

-1

No. of moles

= Mass/MV = 1/66000 = 1.515 x 10-5

1M

Formula for percentage :

E
1cm

E = A/C = 1.177/(1.515x10-5) = 77689.77 M-1cm-1

B)

Adenine A = 0.0517 c= 6.0 mg/l I =261 nm MW : 135.1

6mg = 0.006g( 6g = 6000mg ) 1ml = 0.001I ( 1 I =1000ml ) 6 mg/1 I BSA = 0.006g/1000g What we want is = g/100ml ,so we must (devide 10) Hence.C = 0.0006g/100ml Mass = 0.006g/1000
1%

Formula for percentage :

E
1cm

E = A/C = 0.0517/0.0006 = 861.67 mg-1cm-1 Number of mole = Mass/MW = 0.006/135.1 = 4.44 x 10-5
=0.0517 4.44 x 10 -5 Extinction coefficient ( E ) =1164.4 M-1cm-1

Extinction coefficient ( E )

3) Cytochrome :

A = 0.640M

I =450 nm
=1.0 x10-5 =A

Mole of cytochrome Extinction coefficient ( E ) C Extinction coefficient ( E )

= 0.640 =64000 M-1cm-1

1.0x10-5 Extinction coefficient ( E )

Discussion :

In this experiment, the wavelength that has been used to measure the absorption of bovine serum albumin and adenine are 280 nm and 261 nm respectively. These two wavelengths are in the ultraviolet region. Bovine serum albumin and adenine both are colourless solution. While for cytochrome, visible wavelength ( 450 nm ) used to measure the absorbance. The extinction coefficient of a solution can be used to determine the absorbance of one solution that has different concentration because according to Beer law, the absorbance of substance dissolved in transparent solvent is proportional to the number of absorbing molecules. Conclusion : The absorbance for 1.0 mg / ml of bovine serum albumin at wavelength 280 nm is 0.177 and the extinction coefficient is 77689.77 M-1cm-1 For the 6.0 mg/l of adenine, the absorbance at wavelength 261 nm is 0.0517 and the extinction coefficient is 1164.4. While the extinction coefficient for 1.5 x 10-5 M cytochorme with absorbance 0.640 at wavelength 450 nm is 64000 M-1cm-1

Problem 1

A solution containing 2 mg / l of a light-absorbing substance in a 1 cm cuvette transmits 75% of incident of a certain wavelength. Calculate the transmission of a solution containing: a) 4 mg / liter From question: Log I Log 100 75 0.125 E = Ecl

= E ( 0.002 )( 1 ) = E (0.002 ) = 62.5

For 4 mg / liter Log I Log I I Transmission, T = I I = 1 I Transmission, T = 1 1.778 = 62.5 ( 0.004 )( 1 )

= 0.25

= 1.778

Transmission, T = 0.56

b) 1 mg / liter

Log I Log I I

= 62.5 ( 0.001 )( 1 )

= 0.0625

= 1.155

Transmission, T

1 I

Transmission, T

= 1 1.155

Transmission, T = 0.87 c) 6 mg / liter Log I Log I I Transmission, T = I I = 1 I Transmission, T = 1 2.37 = 62.5 ( 0.006 )( 1 )

= 0.375

= 2.37

Transmission, T = 0.42

d) 5.4 mg / liter

Log I Log I I

= 62.5 ( 0.0054 )( 1 )

= 0.3375

= 2.175

Transmission, T

= I

1 I

Transmission, T

= 1 2.175

Transmission, T = 0.46 e) If the molecular weight of the compound is 250, calculate molar extinction ( Em ) 2 mg / liter Mole of the compound = Transmission, T = 0.002 g / 1000 liter = 0.002 250 8 x 10-6 M = Log I = = Em = = Em = Log 100 75 0.125 A C 0.125 8 x 10-6 15625 M-1cm-1

Principles of Spectrophotometer (2)

Title Objectives : The absorption spectrum. : 1. To determine the absorption spectrum of haemoglobin and absorption spectrum of NADH and NAD+ 2. To calculate the molar absorption coefficient of haemoglobin at the wavelength of maximum absorption Introduction Absorption spectroscopy refers to a wide range of techniques where one measures how much light of a particular wavelength (color) is absorbed by a sample. Since color can often be correlated with the presence and or structure of a particular chemical, absorbance spectroscopy is widely used for both qualitative (is a chemical present) and quantitative (how much) and structural (is it degraded) work in a wide range of fields. For instance, DNA absorbs light in the UV range (which is partly why sunlight is dangerous) so the amount of DNA in a sample can be determined by measuring the absorbance of UV light. The plot of amount of radiation absorbed versus wavelength for a particular compound is referred to as the absorption spectrum. The normalized absorption spectrum is characteristic for a particular compound, does not change with varying concentration and is like the chemical "fingerprint" of the compound. At wavelengths corresponding to the resonant energy levels of the sample, some of the incident photons are absorbed, resulting in a drop in the measured transmission intensity and a corresponding dip in the spectrum. The absorption spectrum can be measured using a spectrometer and by knowing the shape of the spectrum , the optical path length and the amount of radiation absorbed, one can determine the structure and concentration of the compound. 1) Determination of the absorption spectrum of haemoglobin. Method 1. A cuvette was filled with a solution of haemoglobin ( 0.1 mg / ml ) in distilled water.

2. By using the cuvette filled with distilled water, the spectrophotometer reading was set to zero. 3. The absorbance of haemoglobin was measured starting at 400nm up to 680 nm. 4. Then, a graph absorbance against wavelength was plot. 5. The above step was repeated by using solution containing both NADH and NAD+ Result 1) Absorption spectrum of haemoglobin Wavelen gth Absorba nce Waveleng th Absorban ce 400 2.83 4 500 0.63 6 410 2.66 9 510 0.61 9 600 0.35 6 420 2.45 9 520 0.57 7 610 0.35 0 430 1.38 2 530 0.55 0 620 0.34 6 440 0.94 3 540 0.52 5 630 0.34 5 450 0.78 1 550 0.47 9 640 0.31 9 460 0.70 0 560 0.43 2 650 0.26 3 470 0.65 0 570 0.42 0 660 0.22 5 670 0.284 680 0.192 480 0.63 2 580 0.485 590 0.375 490 0.638

Wavelength Absorbance

3 2.5 2
Relative 1.5 A bsorbance

1 0.5 0
40 0 43 0 46 0 49 0 52 0 55 0 58 0 61 0 64 0 67 0
waveleng th in (nm )

The wavelength of maximum absorption of haemoglobin is 400 nm and the value of maximum absorption at this wavelength is 2.834 Molar extinction coefficient ( Em ) 0.1 mg / ml = 1.0 x 10-7 g / l = 65000 6.5 x1011 1.0 x 10-7 g / l Mole of haemoglobin = = A C Em = 2.834 6.5 x1011 Em = 4.36 x 10-12 M-1cm-1

Mole of haemoglobin

Em

2) Absorption spectrum for NADH and NAD+ Wavelength Absorbance 240 2.800 250 3.612 260 3.610 270 3.436 280 1.599 290 0.718 300 0.775 310 1.086

Wavelength Absorbance

320 1.498

330 1.852

340 1.989

350 1.845

360 1.446

370 0.943

380 0.511

390 0.239

4 3.5 3 2.5
R elative 2 Abs orbance

1.5 1 0.5 0
24 0 27 0 30 0 33 0 36 0 39 0
3.612 and 1.989 3) Absorption spectrum for NADH

waveleng th in (nm )

The wavelength at the maximum absorption for NADH and NAD + are 240 nm and 340 nm and the value of maximum absorption at each of these wavelengths are

Wavelength Absorbance

240 0.545

250 0.839

260 1.038

270 0.733

280 0.383

290 0.226

300 0.162

310 0.154

Wavelength Absorbance

320 0.203

330 0.251

340 0.261

350 0.228

360 0.156

370 0.085

380 0.030

390 0.001

The wavelength at the maximum absorption for NADH is 260 nm and the value of maximum absorption at each of these wavelengths is 1.038

1.2 1 0.8
R ela tive 0.6 Absorba nce

0.4 0.2 0
24 0 27 0 30 0 33 0 36 0 39 0
wa veleng thin(nm )
4) Absorption spectrum for NAD Wavelength Absorbance 240 0.307 250 0.400 260 0.442 270 0.278 280 0.086 290 0.036 300 0.030 310 0.023

Wavelength Absorbance

320 0.019

330 0.015

340 0.012

350 0.010

360 0.008

370 0.006

380 0.005

390 0.004

0.5 0.45 0.4 0.35 0.3 R ela tive 0.25 Absorba nce 0.2 0.15 0.1 0.05 0
24 0 27 0 30 0 33 0 36 0 39 0
wa veleng thin (nm )

The wavelength at the maximum absorption for NAD + is 260 nm and the value of maximum absorption at each of these wavelengths is 0.442 Discussion 1) For the absorption spectrum of haemoglobin, the wavelength of maximum absorption is 400 nm and the value of maximum absorption at this wavelength is 2.834 while the wavelength of minimum absorption is 680 nm with the value of absorption 0.192. this result shows that haemoglobin absorb strongly in the blue and green light rays and reflect the red wavelength. NADH and NAD+ are colourless solution. They are absorbs ultraviolet wavelength. They are two peak in the absorption spectrum of NADH and NAD+. These are because NADH and NAD + absorb light in different wavelength. 2) The concenteration of NADH at peak 1 is 3.198 x 10-4 M same as concenteration at peak 2,the concenteration of NAD is -6.583 M the value of

NAD negative because maybe the concenteration of mixture is so high and the concenteration is not equivalent between NAD & NADH. 3) The putting of solution into differente cuvette also infeluent our result if the solution NAD,NADH & Mixture NAD and NADH the wavelengeth that use ( Ultraviolet light ) the solution must put in quartz if the solution is hemoglobin the between 200-400nm

cuvette depend the type of solution

wavelengeth that use between 400-700nm(visible light ) the solution must put in plastic cuvette depend the type of solution Conclusion The peak for absorption spectrum of haemoglobin is at the wavelength 400 nm with absorbance 2.834 and the peaks for absorption spectrum wavelength at the maximum absorption for NADH and NAD + are 240 nm and 340 nm and the value of maximum absorption at each of these wavelengths are 3.612 and 1.989 and for . The wavelength at the maximum absorption for NADH is 260 nm and the value of maximum absorption at each of these wavelengths is 1.038 and the lastly is The wavelength at the maximum absorption for NAD + is 260 nm and the value of maximum absorption at each of these wavelengths is 0.442

Question By referring to the absorption spectrum that you have plotted from ( 2 ) and the value of extinction coefficient give below, calculate the concentration of NAD+ ( oxidize form ) and NADH ( reduce form ) of these coenzyme. E ( M-1cm-1 ) Compounds NAD+ NADH Peak 1 18000 15000 Peak 2 0 6220

The concenteration of NADH(maximum absorption ( Peak 1 = 3.612 , peak 2=1.989 ) Extinction coefficient , E : A/C C = A/E = 1.989/6220 = 3.198 x 10-4 M By using this concenteration,The absorption value for NADH for the first peak can be calculate A ( NADH ) = EC = 15000 x (3.198 x 10-4) = 4.797 The absorption of NAD = A1( Of NAD from graph )-A2( of NADH for above value ) = 3.612-4.797 = -1.185 From here we can know the NADH has 2 peak that shown on mixture graph (intermediate curve) between NAD and NADH & The NAD absorbance value is lowes ( represented by negative absorbance value)

Concentration of NAD : CNAD = A/E = -1.185/18000 = -6.583 M Answer from the other group that get a positive answer Concentration of NADH : E = 6220 M-1cm-1 A = 0.39 C=A E = 0.39 6220 = 6.27 x 10-5 M Using this value, the absorbance value for NADH for the first peak can be calculated A (of NADH) = EC = 1500 x ( 6.27 x 10-5 ) = 0.9405 The absorbance of NAD+ = A( from the first peak ) - A(of NADH from the above value ) = 1.18 0.9405 = 0.2395 So, from here we can calculate the concentration of NAD + C=A E = 0.2395 18000 = 1.33 x 10-5 M