Methods In Clinical Chemistry- Centrifugation, Spectrophotometry, Flame photometry, Ion-Selective electrodes, Chromatography

Question 1

_____ is a basic separation technique

Answer 1

Centrifugation

Question 2

Centrifugation is a process in which centrifugal force is used to separate ______ from _______

Answer 2

solid matter from a liquid suspension

Question 3

The centrifuge consists of

Answer 3

a head or rotor, carriers, or shields that are attached to the vertical shaft of a motor and enclosed in a metal covering.

Question 4

Some centrifuges include a brake or a built in tachometer, which indicates ?

some centrifuges are refrigerated.

Answer 4

Speed

Question 5

Centrifugal force depends on three variables:

Answer 5

mass,
speed,
and radius.

Question 6

The speed is expressed in

Answer 6

revolutions per minute (rpm)

Question 7

the centrifugal force generated is expressed in terms of

Answer 7

relative centrifugal force (RCF) or gravities (g)

Question 8

The speed of the centrifuge is related to the RCF by the following equation:

Answer 8

RCF = 1.118 x 10r-5 x r x (rpm)squared

where 1.118 x 10-5 is a constant, determined from the angular velocity, and r is the radius in centimeters, measured from the center of the centrifuge axis to the bottom of the test-tube shield

Question 9

Centrifuge classification is based on several criteria, including

Answer 9

benchtop or floor model,
refrigeration,
rotor head (e.g., fixed, hematocrit, swinging-bucket, or angled),
or maximum speed attainable (i.e., ultracentrifuge).

Question 10

Centrifuges are generally used to separate 3 things. These are

Answer 10

(1) serum or plasma from the blood cells as the blood samples are being processed;

(2) separate a supernatant from a precipitate during an analytic reaction;

(3) separate two immiscible liquids, such as a lipid-laden sample; or to expel air

Question 11

Centrifuge care includes

Answer 11

daily cleaning of any spills or debris, such as blood or glass,

and ensuring that the centrifuge is properly balanced and free from any excessive vibrations.

Question 12

Balancing the centrifuge load is critical. What happens when centrifuge load is unbalanced?.

Answer 12

Many newer centrifuges will automatically decrease their speed if the load is not evenly distributed, but more often, the centrifuge will shake and vibrate or make more noise than expected

Question 13

The centrifuge cover should remain closed until the centrifuge has come to a complete stop to avoid any ___________

The specific manufacturer’s service manual should be consulted for details on how to change brushes and on lubrication requirements.

Answer 13

aerosol contamination

Question 14

The speed of a centrifuge is easily checked using a________or ________ . The hole located in the lid of many centrifuges is designed for speed verification using these devices but may also represent an aerosol biohazard.

Accreditation agencies require periodic verification of centrifuge speeds.

Answer 14

tachometer or strobe light

Question 15

A centrifuge needs to be balanced based on equalizing both the volume and weight distribution across the centrifuge head.

Many laboratories will make up ___________that approximate routinely used volumes and tube sizes, including the stopper on phlebotomy tubes, which can be used to match those needed from patient samples.

Answer 15

balance” tubes

Question 16

The majority of techniques fall into one of four basic disciplines within the field of analytic chemistry:

Answer 16

spectrometry (including spectrophotometry, atomic absorption, and mass spectrometry);

luminescence (including fluorescence, chemiluminescence, and nephelometry);

electroanalytic methods (including electrophoresis, potentiometry, and amperometry);

chromatography (including gas, liquid, and thin-layer)

Question 17

SPECTROPHOTOMETERS AND PHOTOMETERS measure

Answer 17

electromagnetic radiation passing through a solution.

Question 18

Difference between photometer and spectrophotometer

Answer 18

Photometers measure light intensity without consideration of wavelength
Spectrophotometers use filters to select (isolate) a narrow range of the incident wavelength

Question 19

Radiant energy that passes through an object will be _____, _______& _______

Answer 19

partially reflected,
absorbed,
and transmitted.

Question 20

Electromagnetic radiation is described as

Answer 20

photons of energy traveling in waves.

Question 21

The relationship between wavelength and energy E is described by

Answer 21

Planck’s formula: E= hv

where h is a constant (6.62x 10-27 ), known as Planck’s constant, and v is frequency.

Question 22

Because the frequency of a wave is inversely proportional to the wavelength, it follows that the energy of electromagnetic radiation is _______ proportional to wavelength

Answer 22

inversely

Question 23

Electromagnetic radiation include

Answer 23

Cosmic
Gamma rays
X-rays
Ultraviolet
Visible
Infra red
Microwaves( radio, tv, radar)

Question 24

Visible light falls in between, with the colour _________ and _________ wavelengths being the approximate limits of the visible spectrum.

Answer 24

violet at 400-nm and red at 700- nm

Question 25

T he spectrophometer and the flame photometer measure

Answer 25

absorption of radiant energy to determine concentration of atoms or molecules.

Question 26

T he relationship between absorption of light by a solution and the concentration of that solution has been described by

Answer 26

Beer and colleagues

Question 27

Beer’s law states that

Answer 27

the concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light.

Question 28

% transmittance

Answer 28

% transmittance = transmitted/incident x 100

Question 29

Percent transmittance is the ratio of the radiant energy transmitted (T) divided by the radiant energy incident on the sample (I).

if all light is absorbed it will result in

Answer 29

0% T.

Question 30

A level of 100% T is obtained if

Answer 30

no light is absorbed.

All is transmitted

Question 31

The electrical readout of the instrument is set arbitrarily at 100% T, while the light is passing through a “

Answer 31

blank” or reference

Question 32

The sample containing absorbing molecules to be measured is placed in the light path. The difference in amount of light transmitted by the blank and that transmitted by the sample is due only to

Answer 32

the presence of the compound being measured.

Question 33

The % T measured by commercial spectrophotometers is the ratio of

Answer 33

the sample transmitted beam divided by the blank transmitted beam.

Question 34

As concentration of the sample increases, % T decreases in a ______ manner.

Answer 34

Logarithmic

Question 35

Absorbance A is the amount of light absorbed. It cannot be measured directly by a spectrophotometer but rather is mathematically derived from % T as follows: From Beer’s law, absorbance is_____ proportional to the logarithm of the transmitted light.

Answer 35

inversely

Question 36

Formulation of formula

Question 37

According to Beer’s law, absorbance is directly proportional to concentration.

Answer 37

A ∞ C
A = e x b x C
e and b are constants
where e molar absorptivity; the fraction of a specific wavelength of light absorbed by a given type of molecule; b is the length of light path through the solution; and C is the concentration of absorbing molecules.

Question 38

Therefore A/C = constant
So for a given set of reaction,

Answer 38

A1/C1 =A2/C2

Question 39

A spectrophotometer is used to measure

Answer 39

the light transmitted by a solution in order to determine the concentration of the light-absorbing substance in the solution

Question 40

Components of a spectrophotometer

Answer 40

Light source
Monochromators
Sample cell /cuvet
Photodetector

Question 41

Light source

Answer 41

The most common source of light for work in the visible and near-infrared region is the incandescent tungsten or tungsten-iodide lamp.
The lamps most commonly used for ultraviolet (UV) work are the deuterium-discharge lamp and the mercury-arc lamp.

Question 42

Monochromators

Answer 42

Isolation of individual wavelengths of light is an important and necessary function of a monochromator.

The degree of wavelength isolation is a function of the type of device used and the width of entrance and exit slits.

Numerous devices are used for obtaining monochromatic light. The least expensive are coloured-glass filters. You also have interference filters, glass prisms and diffraction gratings etc

Question 43

Sample cell

Answer 43

The next component of the basic spectrophotometer is the sample cell or cuvet, which may be round or square.

The light path must be kept constant to have absorbance proportional to concentration

Cuvets with scratched optical surfaces scatter light and should be discarded.

Inexpensive glass cuvets can be used for applications in the visible range, but they absorb light in the UV region.

Quartz cuvets must, therefore, be used for applications requiring UV radiation.

Question 44

Photodetectors

Answer 44

The purpose of the detector is to convert the transmitted radiant energy into an equivalent amount of electrical energy.
The least expensive of the devices is known as a barrier-layer cell, or photocell.
The photo cell is composed of a film of light-sensitive material, frequently selenium, on a plate of iron.
Over the light-sensitive material is a thin, transparent layer of silver. When exposed to light, electrons in the light-sensitive material are excited and released to flow to the highly conductive silver.
The produced current is proportional to incident radiation.
Digital signals are processed electronically to produce absorbance readings.

Question 45

What happens in flame optometry

Answer 45

The flame-emission photometer, which measures light emitted by excited atoms, was widely used to determine concentration of Na, K, or Li.
With the development of ion selective electrodes for these analytes, flame photometers are no longer routinely used in clinical chemistry laboratories.

Question 46

In ion selective electrodes,

Answer 46

Potentiometric methods of analysis involve the direct measurement of electrical potential due to the activity of free ions.
Ion-selective electrodes (ISEs) are designed to be sensitive toward individual ions.
An ISE universally used in the clinical laboratory is the pH electrode
The basic components of a pH meter are the indicator electrode, the reference electrode, the liquid junction and the read-out meter.

Question 47

Components 1

Answer 47

(1) The indicator electrode : The indicator electrode of a pH meter consists of a silver wire coated with AgCl, immersed into an internal solution of 0.1 mmol/L HCl, and placed into a tube containing a special glass membrane tip.
This membrane is only sensitive to hydrogen ions (H).
Glass membranes that are selectively sensitive to H consist of specific quantities of lithium, cesium, lanthanum, barium, or aluminum oxides in silicate

When the pH electrode is placed into the test solution, movement of H near the tip of the electrode produces a potential difference between the internal solution and the test solution

Question 48

2 The reference electrode

Answer 48

Reference electrodes generally consist of a metal and its salt in contact with a solution containing the same anion
All reference electrodes must generate a stable electrical potential.

The reference electrode commonly used is the calomel electrode.
Calomel, a paste of predominantly mercurous chloride, is in direct contact with metallic mercury in an electrolyte solution of potassium chloride.
As long as the electrolyte concentration and the temperature remain constant, a stable voltage is generated at the interface of the mercury and its salt

Question 49

3 the liquid junction

Answer 49

Electrical connection between the indicator and reference electrodes is achieved by allowing a slow flow of electrolyte from the tip of the reference electrode.
A junction potential is always set up at the boundary between two dissimilar solutions because of positive and negative ions diffusing across the boundary at unequal rates.

Electromotive force produced by the reference and indicator electrodes in the test solution is in the millivolt range and read by

Question 50

4?

Answer 50

(4) The Readout meter and converted to concentration units using the Nernst equation.
In the Clinical Chemistry lab, the ISE is used to determine the concentration of ions such as Na, H, K, Ca, HCO3, CL, etc