Spectrophotometry and Plotting of Calibration Curve BIO-2

PURPOSE To understand the principles of spectrophotometry To understand the structure of spectrophotometer To understand the standard calibration curve and determine the concentration of CuSO 4

What is the function of spectrophotometry? What is spectrophotometry? Why do we use it? How does it work? Questions

Spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. A spectrophotometer is employed to measure the amount of light that a sample absorbs. The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector. What is spectrophotometry? What is the function of spectrophotometer?

Polychromatic light polychromatic light composed of more than one wavelength, having or exhibiting many colors. Polychromatic light Monochromatic light light monochromatic light Light of one color, having wavelengths confined to an extremely narrow range.

SPECTRUM Spectral Distribution of Radiant Energy 380nm ~ 760nm Infrared(IR) Ultraviolet(UV)

t Ia Ir Why do you know some light is absorbed by solution?

Complementary color Colour of substanceColour of absorbed light Wavelengt of absorbed light olivine Yellow Orange red Reddish violet violet Blue greenish-blue blusih green violet Blue greenish-blue Green Olivine Yellow Orange red 380~435 nm 435~480nm 480~500nm 500~560nm 560~580nm 580~595nm 595~650nm 650~760nm Solution can absorb light selectly.

图 1-2 KMnO 4 溶液的吸收曲线 A λ(nm ) Wavelength of Maximal Absorption(525nm) Potassium permanganate

Transmittance = T = (I t / I 0 ) ×100% Absorbance= A = ? A=-lgT=lg(I 0 /I t ) Transmittance and Absorbance When a ray of monochromatic light of initial intensity (I o ) passes through a solution in a transparent vessel, some of the light is absorbed (I a ) so that the intensity of the transmitted light (I t ) is less than I o. ItIt IaIa I0I0

As the cell thickness increases, I t (transmitted intensity of light ) decreases. LAWS OF ABSORBTION OF LIGHT ItIt

 Lambert’s law: length-dependent I = I o e -kL or A=kL Where ‘k’ is a constant, e = base of natural log L= length of the light path in the vessel.  Beer’s law: concentration-dependent I = I o e -kC or A=kC Where ‘k’ is constant and ‘c’ = concentration solution. Combining both Lambert’s - Beer’s law, we have: I = I o e -kLC or A=kLC

A =-lgT= k L C k: extinction coefficient L: length of the light path C: concentration

Blank: This will help to exclude the absorption due to reagents. Standard: it includes a solution of known concentration of the substance which is going to be determined in the test container. Test: it contains an unknown quantity of the substance. When determinations are made, one must be sure that the absorption produced is due to the particular substances, not by the solvent and compounds in the reagents. The batch of analysis must include the following solutions.

Standard contrast method Let the conc. of standard = C 1, and absorbance = A 1 So, A 1 = klC 1 Let the conc. of unknown = C 2, and absorbance = A 2 So, A 2 = klC 2 So, A 1 /A 2 = klC 1 / klC 2 Or, C 2 = [A 2 /A 1 ] x C 1 C test = [A test /A standard ] x C standard Calculate Conc. of unknown

There is some A vs. C where graph is linear. Avoid very high or low absorbencies when drawing a standard curve. The best results are obtained with 0.1 < A < 1. Plot the Absorbance vs. Concentration to get a straight line. NEVER extrapolate beyond point known where becomes non-linear. Standard Curve method

Structure of Spectrophotometer

Spectrophotometry Spectrophotometer Sample Cuvette Sample room Cuvette holder

Spectrophotometer Adjust Wavelength Pull Rod of Cuvette Show Wavelength Sample Room Mode On/Off 100%T/0A 0%T

How to operate Spectrophotometer ? 1. Turn on ， set wavelength ， warm-up for 20min 2. Respectively move sample solutions to cuvettes Blank, Standard, Test Height: 2/3~4/5 Hold the rough face ， keep the smooth face tidy. Put cuvettes into the cuvette holder in the proper order. Blank Standard Test1 Test2 cuvette holder

3. To “Blank”, mode “T”, press “100%T/0A”, Set T =100 or A=0. 4. Pull the pole once time, press “0%T”, Set T =0. 5. Repeat step “3” to “4”. 6. Change mode to “A”. 7. pull the pole second time, record A1; Third time,record A2; Forth time,record A3. Operating steps of Spectrophotometry

Blank Standard Test1 Test2 Blank Standard Test1 Test2 Blank Standard Test1 Test2 Blank Standard Test1 Test2 Blank Standard Test1 Test2 T handle 0 set T 100% ： Blank, A=0 T handle 1 set T 0% ： A= A handle2 、 3 、 4 assay A ： T est 1 、 2 、 3 Determine Mode: T 0% ~ 100% A 0 ~ ∞

CuSO 4 x% = ? Methods: 1. Standard curve 2. Standard contrast

Reagents & Materials 5% CuSO 4 X% CuSO 4 dH 2 O Test tubes Pipettes Spectrophotometer Cuvette

1. Standard curve method Num5%CuSO 4 (ml)H 2 O(ml)C(%)A X%CuSO Method Mix the contents of each tube, measure the absorbance(A) of each tube at 650nm, setting zero with distilled water.

C1C2C3C4C5 A1 A2 A3 A4 A5 Standard curve

2. Cuso4 (x%) 5ml, Ax Ax Cx X% CuSO 4 : Ax=? Cx=?

2. Standard contrast method A s = k LC s A x = k LC x x% CuSO 4, A x =? 5%CuSO the standard, A s =? C x =? Calculation:

Discussion Compare the two methods and the results, which one is better? why?