بسم الله الرحمن الرحيمUMM AL-QURA UNIVERSITYFaculty of MedicineDep. Of BIOCHEMISTRY

INSTRUMENTS

• For student to be familiar with the use of the following:

1. Pipettes and pipetting techniques.2. pH meter.3. Spectrophotometer.

OBJECTIVES:OBJECTIVES:

Volumetric (Bulb) pipettes: These are designed to deliver the exact

amount of solution. Has only one point of graduation.

Graduated pipettes: Which are graduated along the stem, but

not at the tip. They are less accurate than the volumetric

pipettes, but can deliver small fractions of the total capacity of the pipette.

1 -Pipettes:

Other types of pipettes:• These include the micropipette, which are

used to measure small quantities that the regular pipettes cannot measure, i.e. micro litter amounts (ul.).

Fixed volume pipette: This delivers only the exact volume which

is written on the top of the pipette (e.g. 20 ul, 100 ul, 1000 ul, … etc.).

Adjustable or variable volume pipette:

This can be calibrated to deliver any amount of solution which is written on the side of the pipette

Use of pipettes:1. Never use mouth, use pipette bulbs or

pipette filters in aspirating solutions.2. Pipettes must be clean always.3. After aspirating the required volume, wipe

off the surface of the pipette with tissue paper.

4. Always read the bottom of the meniscus for clear solutions, and top for colored or viscous ones.

5. The graduation point and your eyes should be in a horizontal position.

6. When delivering solutions, let the tip of the pipette touch the inner surface of the container, and let the solution flow free.

7. Always choose the proper type of pipette in measuring the required volume.

Beakers: Available in different volumes .

Conical Flasks: Used in titration procedures and other different uses, also available in different volumes.

Graduated Cylinders:For measuring the volume of solutions and dilution procedures.

Burette:For addition of volumes in titration

procedures.

2 -pH meter

The pH meter is an instrument that is used to measure the pH (Acidity or alkalinity) of solutions.

It is composed of the following:1. Glass-bulb electrode.2. Reference electrode.3. Sensitive meter or measuring

device.

pH measurement procedure:

• Leave the pH meter in STD BY mode to eliminate warm up and increase component life. Allow one half hour warm up if meter has been disconnected.

• Securely connect pH and reference electrodes, or combination pH electrode into INPUT (pH) on the rear panel.

• Verify that reference filling solution level in the electrode is adequate. The level of filling solution should be higher than the sample level.

• Stir buffer and sample during analysis with a magnetic stirrer, if possible.

• Rinse electrodes with distilled water between measurements.

3-spectrophotometer• Spectrophotometer is one of the

most commonly used instruments in the biochemistry laboratory.

• Its main function is to measure the absorbance or concentration of substances.

Beer-Lambert Law:

• Beers Law states that: when a ray of monochromatic light passes through an absorbing medium, the intensity decreases as the concentration of the absorbing medium increases.

• Lamberts law states that: when a ray of monochromatic light (single wavelength) passes through an absorbing medium, its intensity decreases as the length of the medium increases.

• These two laws are combined in the form of Beer-Lambert Law and expressed as: A = abc

• Where: A= Absorbance. a= Extension coefficient (constant), which is

defined as the absorbance (at a given wavelength) of 1M solution of the pure substance through a 1cm. Light path.

b= Length of light path. = 1cm. c= Concentration of the substance in the

solution.

components of the spectrophotometer:

• i) Light source:• This part of the instrument emits visible or

UV light depending on the source itself. For example tungsten lamp emits light from 400-750 nm, while a deuterium lamp emits light in the UV region.

• ii) Monochromator:• This part is to a prism or a filter which

functions in isolating only one single light. i.e. one wavelength.

• iii) Slit:• This part functions in passing a very fine

beam of isolated wavelength.

components of the spectrophotometer:• iv) Cuvette holder:• This is where the sample of the colored solution is

inserted. The cuvette can be either in the round shape like a test tube or square form. When measuring absorbance in the visible region the cuvette is usually made of glass, while in measuring absorbance at the low UV region cuvettes are made of quartz.

• v) Photo cell:• This part converts light energy to electric energy so

it can be measured by the measured by the measuring device.

• vi ) Galvanometer:• This is where the electric pulses are received and

converted on a scale either to absorbance or to transmittance units.

ABDULLATIF TAHA ABDULLAMSc. BIOCHEMISTRY