Biochem Lab Spectro and Centrifuge (Buhay ay di karera)

What is centrifugation? What particles typically become sediments?

The spinning of stuff at high speed (generation of centrifugal force), to separate components of a mixture by means of their size, shape, or viscosity of a medium, and density of a particle.

Denser particles

What is centrifugal force?

the apparent force pushing a body outward from the center of a curved path, but it is a fictitious force, not a real one, experienced in a rotating reference frame. It is the result of inertia, where the object's tendency to move in a straight line feels like an outward push.

Principles of centrifugation

Can be refrigirated depending on sample stability and machine model to prevent sample degredation

can work under vacuums typically those who reach high speeds

label the parts

1. Latch

2. Chamber

3. Lid

4. Rotor

5. Motor

6. Control

What do you call the rotating part of that holds the tubes and spins?

Rotor

What do you call the part that powers the rotor to generate centrifugal force?

Motor

What are the containers that securely hold on to the sample in the rotor?

Sample Holder/Buckets

What connects the rotor to the motor to transfer rotational force?

Spindle

The part of the machine that allows users to set parameters like speed, time, and temperature?

Control Panel

Part of the machine that Maintains low temperatures during operation in refrigerated centrifuges?

Refrigeration System

The part of the machine that covers the chamber and prevents operation or opening during spinning?

Lid and Safety Lock

What is the outer shell that protects internal components and supports the device?

Chassis or Housing

Two classifications according to rotor

• Horizontal / Swing-Bucket Rotor

• Fixed-Angle Rotor

What is a Horizontal/ Swing Bucket Rotor and its functions?

Buckets swing out horizontally during spinning, ideally used for the separation of multiple components according to density (density-gradient centrifugation)

This method is ideal for separating multiple components in a single sample, such as blood cells, organelles, or DNA, as it produces a sharp and well-defined separation.

What is a Fixed-Angle Rotor and its functions?

Holds tubes at a fixed angle, suitable for fast pelleting of particles

This type of rotor is particularly effective for collecting a small, dense pellet from a large volume of liquid, such as bacteria, yeast, or cell debris.

What is RPM (revolutions per minute)?

– The speed at which the rotor spins and is also the number of times the centrifuge rotor spins in one minute. It's a straightforward measure of rotational speed.

What is RCF (relative centrifugal force)?

the force applied to a sample during centrifugation, expressed as a multiple of the Earth's gravitational force (g)

What determines how quickly the rotor speeds up or slows down, allowing gradual acceleration/deceleration to protect fragile samples and prevent tube damage?

Acceleration/Deceleration Ramp

Typical units of time and temperature

Time= Minutes

Temp.= Celsius

Do’s in operating centrifuge?

• Use the appropriate centrifuge tube

• Pre-cool centrifuge and rotor if applicable

• ensure the correct rotor is used and installed properly

• Balance the load in the rotor

Don’ts in using a centrifuge

• Leave the centrifuge unattended if not at maximum speed or running smoothly

• Ignore the safety features

What are colorimetric assay?

the process that measure the light absorbed by the substance, in the visible or UV region of the electromagnetic spectrum, using an ultraviolet-visible (UV-Vis) spectrophotometer

What is spectrophotometry

used to measure the amount of light transmitted through a sample at a given wavelength

The colors of the visible light spectrum

Part of the spectrophotometer that  Emits a broad range of light used to illuminate the sample

Light Source

Part of the spectrophotometer that selects a specific wavelength of light from the white light source

Monochromator

Part of the spectrophotometer that holds the sample in a transparent container for light to pass through

sample compartment (cuvette)

Part of the spectrophotometer that measures transmitted light and displays the corresponding absorbance or transmittance

Detector and Readout

What is the formula for transmittance?

Incident light (before) / Transmitted light (after)

what is the formula for absorbance?

Log 10 (Transmitted (after) / Incident (after) )

Most commonly used type of spectrophotometer in biochemical analysis?

microplate spectrophotometer

Describe the relationship of absorbance and solution concentration

Linear / Direct

proportion/solution used to estimate the concentration of the unknown given a known concentration

A over C

What is a Spectrophotometric Calibration Curve and how is it used to determine the unknown concentration of a substance?

also known as a Standard Curve is a graph that helps you find the concentration of an unknown sample. You first create the curve by measuring the absorbance of several solutions with known concentrations. You then plot these absorbance values (y-axis) against their corresponding concentrations (x-axis) and draw a straight line through the points.

Picture ni Master Yoda