Principles of Scientific Instrumentation

Design of Experiments and Analysis of Results

80-801

Bldg 102 Room 3

12:00-14:00 ThursLecturers:

Dr. Alexander Varvak

Dr. Chaim Wachtel

Dr. Refael Minnes

Dr. Itay Lazar

Structure of the course

• Weekly lectures• Requirements: Course is geared for MS/PhD

students who conduct experimental research in Biological Sciences

• Course materials can be found at

http://lifefaculty.biu.ac.il/FacultyInstruments/• Grade is based on the class presentation

(date to be determined)

Preparation of Buffers - 1

Calculate the volume of sulfuric acid (H2SO4) necessary to prepare 600 milliliter 0.5M H2SO4 from concentrated H2SO4 stock (assume 100%).MW H2SO4: 98.1 g/molDensity H2SO4: 1.84 g/cm3

Calculation:0.5M H2SO4 x 98.1 g/mol = 49.05 g/liter 49.05 g/liter x 0.6 L = 29.43 g H2SO4

29.43 g H2SO4 / 1.84 g/mL = 15.99 mL H2SO4

ALWAYS ADD ACID TO WATER!!!Take 550-580 mL water, add 16 mL concentrated sulfuric acid, then add water to 600 mL

Preparation of Buffers - 2

Preparation of monocomponent buffer stocks. Given:

MW of Na2HPO4 ∙ 12H2O = 358.14 g/mol

MW of Na2HPO4 ∙ 2H2O = 177.99 g/mol

(a)Calculate the weight of dibasic sodium phosphate dodecahydrate (Na2HPO4 ∙ 12H2O) powder required to prepare 1 liter of 1M stock of Na2HPO4 solution.

(b)Calculate the weight of dibasic sodium phosphate dihydrate (Na2HPO4 ∙ 2H2O) required to prepare the same solution as in (a).

Preparation of buffers - 3

Monobasic potassium phosphate has pKa of 7 at room temperature. To prepare 1 liter of 0.5M potassium phosphate buffer at pH 7.5 by mixing stocks of 0.5M monobasic potassium phosphate (pH 4.5) and 0.5M dibasic potassium phosphate (pH 9.5), you will need approximately (choose the best answer):

A. 500 mL of each solutionB. 333 mL of monobasic solution and 667

mL of dibasic solutionC. 667 mL of monobasic solution and 333 mL of dibasic solution

Preparation of Buffers - 4

Preparation of glucose solution.• Density of water = 1 g/mL

• Solubility of glucose: 91g in 100 mL of water

• Density of glucose 1.54 g/mL

In order to prepare 100 mL of 50% (weight / vol) solution of glucoseA.Mix 50 g glucose with 50 mL of water

B.Mix 50 g glucose with 100 mL of water

C.Mix 50 g glucose enough water to dissolve it completely, then add water to 100mL total volume.

Preparation of Buffers - 4

1. What is the volume of 100 g of 50% weight/weight solution of glucose in water at room temp (25C)?

In principle, it would be calculated as follows:

V(H2O) = (50 g) x (1 mL/1 g) = 50 mLV(glucose) = (50 g) x (1 mL/1.54 g) = 32.5 mL

Total Volume = V(H2O) + V(glucose) = 82.5 mLBUT: At room temp, 50 g of glucose will not

dissolve in 50 mL of water (solubility exceeded, 45 g will dissolve only)

Scientific Method

2. Hypothesis

3. Experiment4. Theory

1. Question/Observation

History of Instrument Use

Light

electromagnetic radiation

Visible Light Spectrum

colour region wavelength (nm)

violet 380 - 435

blue 435 - 500

cyan 500 - 520

green 520 - 565

yellow 565 - 590

orange 590 - 625

red 625 - 740

In 1900, Max Planck developed a new theory of black-body radiation that explained the observed spectrum

A photon has an energy, E, proportional to its frequency, f, by

where h is Planck's constant, λ is the wavelength and c is the speed of light

E

E hhc

Electron orbital transitions

Characteristic UV-vis absorption spectrum

absorbance A (also called optical density) is defined as

Absorbance

A = log10 I0 / I

Transmission

T = I / I0

%T = 100 T

Beer Lamert’s Law

Relationship between A(OD) and %T

Transmittance, T = P / P0%

Transmittance, %T = 100 T

Absorbance, A = log10 P0 / PA = log10 1 / T A = log10

100 / %TA = 2 - log10 %T 

Polarization

Reflection

Light scattering

reflection

scattering

For Solution: Scattering 1/4

Prism

Diffraction grating

Spectrophotometer types -Single beam-Dual beam-Diode array

Single Beam - Spectrophotometer

Dual Beam - Spectrophotometer

Dual Beam – Single Detector

Diode Array - Spectrophotometer

NanoDrop