Spectrophotometry and Plotting of Calibration Curve BIO-2 Spectrophotometry and Plotting of Calibration Curve

PURPOSE To understand the principles of spectrophotometry To understand the structure of spectrophotometer To understand the calibration curve and determine the concentration of CuSO4

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

What is spectrophotometry? Spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. What is the function of spectrophotometer? 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.

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

SPECTRUM In physics, the light refers to electromagnetic wave, it exhibits properties of both wave nature and particle nature. Spectral Distribution of Radiant Energy 380nm ~ 760nm Infrared(IR) Ultraviolet(UV)

Why solutions appear different color? When the white light (the polychromatic light) strikes the object, different objects appear different color due to the particular wavelength of light they absorbed. When the white light strike a colored solution, certain wavelengths can be absorbed and the remainder transmitted. The observed color is the color to be transmitted.

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

A Wavelength of Maximal Absorption(525nm) Absorption spectrum of potassium permanganate (KMnO4) at four different concentrations The different concentration of potassium permanganate solutions have similar spectrum and same λmax. The absorbance will increase if the concentration increase, which can be used as the basis for quantitative analysis.

Transmittance and Absorbance Ia I0 When a ray of monochromatic light of initial intensity (Io) passes through a solution in a transparent vessel, some of the light is absorbed (Ia) so that the intensity of the transmitted light (It) is less than Io. Transmittance = T = (It / I0) ×100% Absorbance= A = ? A=-lgT=lg(I0/It)

If all the light passes through a solution without any absorption, then T=100%, and A=0. If all the light is absorbed without any transmittance, then T=0, and A is infinite.

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

LAWS OF ABSORBTION OF LIGHT Lambert’s law: length-dependent I = Io 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 = Io e-kC or A=kC Where ‘k’ is constant and ‘c’ = concentration solution. Combining both Lambert’s - Beer’s law, we have: I = Io e-kLC or A=kLC

Lambert-Beer law k: absorption coefficient L: length of the light path A =-lgT= k L C k: absorption coefficient L: length of the light path C: concentration The absorption coefficient is the characteristic constant of the subjects.

The choice of conditions of determination The choice of wavelength It is observed that at the wavelength of maximum absorbance (λmax) the deviations is minimal and the sensitive is higher. Due to this reason we generally select λmax for a given sample to measure the absorbance.

The choice of the range of absorbance measurement It is proved by theory that when T=36.8%（or A=0.434）the relative error of concentration is smallest. To minimize the error, T must control to 20~65% (or A= 0.2~0.7). So when design the experiment, you must choose appropriate amount of sample and proper type of cuvette to make the absorbance among this range.

Blank: this will help to exclude the absorption due to reagents. 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. 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.

Standard Contrast method Calculate Conc. of unknown Let the conc. of standard = C1, and absorbance = A1 So, A1 = klC1 Let the conc. of unknown = C2, and absorbance = A2 So, A2 = klC2 So, A1/A2 = klC1 / klC2 Or, C2 = [A2/A1] x C1 Ctest= [Atest/Astandard] x Cstandard

Standard Curve method 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.

Structure of Spectrophotometer

Spectrophotometry Spectrophotometer Sample room Cuvette holder Sample Cuvette

Spectrophotometer Display lamp Of Mode Show Wavelength Sample Room Pull Rod of Cuvette Mode (A, T) 100%T/ 0A 0%T Adjust Wavelength Show Wavelength Display lamp Of Mode

How to operate Spectrophotometer ? 1. Turn on ，set wavelength ，warm-up for 20 min. 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. Test2 Test1 Standard Blank (Notice: Smooth side face to the light). cuvette holder

Operating steps of Spectrophotometry 3. Set “Blank solution” face to the light, adjust Mode to “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.

Determine Blank Standard Test1 Test2 Blank Standard Test1 Test2 Blank Set T=0 Blank Standard Test1 Test2 Set T=100 Determine T handle 0 set T 100% ： Blank, A=0 T handle 1 set T 0% ： A=1.----- A handle2、3、4 assay A： Test 1、2、3

Determine the concentration of unknown solution by spectophotometry CuSO4 x% = ? Methods: 1. Standard curve 2. Standard contrast

Reagents & Materials 5% CuSO4: dissolved 5 g anhydrous CuSO4 in 100 mL distilled water. X% CuSO4 dH2O Test tubes Pipettes Spectrophotometer Cuvette

Method 1. Standard curve method Num 5%CuSO4 (ml) dH2O(ml) C(%) A 1 1.00 4.00 2 2.00 3.00 3 4 5 5.00 0.00 6 X%CuSO4 Mix the contents of each tube, measure the absorbance (A) of each tube at 650nm , setting zero with dH2O.

Standard curve A1 A2 A3 A4 A5 C1 C2 C3 C4 C5 The graph needs to be a best-fit straight line.

2. Cuso4 (x%) 5ml , Ax Ax Cx X% CuSO4: Ax=? Cx=?

2. Standard contrast method Calculation: 5%CuSO4 --- the standard, As=? x% CuSO4, Ax=? As = k LCs Cx=? Ax = k LCx

Discussion Compare the two methods and the results, which one is better? why? Why determine the absorbance of CuSO4 solution at 650 nm? If the absorbance of unknown CuSO4 solution above the range of standard curve, how to determine the concentration of this CuSO4 solution?