QUIZ 1A LEC Question 6

Question 1 A device for rapid pipetting made of spring activated

plunger in a precision bore with disposable tip
How close a measurement to the true value is called
o Volumetric pipet
o Precision
o Micropipette
o Significant
o Serological pipette
o Accuracy
o Dropper
o Estimate

Question 2
Question 7
A substance of known composition, value of which is
established by an analytical procedure Precision pipet considered most accurate is (di ko sure
potek. Kasi sabi diba pinakaccurate yung volumetric eh
o Reagent
to deliver ang volumetric. Tapos basta hindi Ostwald
o Blank
sagot sa isa)
o Control
o Standard o Ostwald-Folin pipet
o Serological pipet
o TC volumetric pipet
Question 3
o TD volumetric pipet
The chemical reagent that has the highest purity for
laboratory use
Question 8
o USP
In the proper use of a volumetric pipet calibrated to
o Analytical grade
deliver (TD) one should
o Reagent grade
o Technical grade o Blow out the solution immediately
o Drain the content but do not blow out
o Wipe the outside wall of the pipet
Question 4 o Rinse the content several times to mix the
solution
Glassware of choice for dilution and preparation of
solutions that are made to definite volume

o Volumetric flask Question 9

o Graduated cylinder
Plastic wares are useful in the laboratory because of
o Erlenmeyer flask
what characteristic(s)?
o Automatic pipet
o All options are correct
o Shatter proof
Question 5
o High impact and tensile strength
A one gram equivalent weight of the solute in one liter o Chemically inert
of solution is expressed as

o One normal solution

o One percent solution
o One molar solution
o One milliequivalent per liter
Question 10 Question 15

Solutions that are used in keeping the pH relatively It has no markings down to the tip allows the content of
constant the pipet to be blown out

o Standards o Serological pipet

o Buffers o To deliver pipet
o Enzymes o Mohr pipet
o Water o Transfer pipet

Question 11 Question 16

One gram equivalent weight of an element equals the Grades of chemicals include all of the following except
gram molecular weight divided by the
o Commercial grade
o Valence o Industrial grade
o Volume o Analytic reagent
o Dilution o Chemically pure
o Mole o

Question 17

Question 12 Water from which minerals have been removed is called

A molar solution contains o Charcoal activated water

o Reagent grade water
o One gram equivalent weight of solute per liter
o Distilled water
of solution
o Deionized water
o None of the options are correct
o One gram molecular weight of solute per liter of Question 18
solution
When should the sides of the pipet be wiped off?
o One gram of solute to 99 gram of solvent
o After reaching the lower meniscus of the mark
o Only when using TC or TD pipet
Question 13
o When using serological pipet and pipette
What happens to a decimal place in the number when o Before lowering the meniscus to the calibrated
you are converting from a smaller unit to a larger unit? mark

o Converting doesn’t require placing

o The decimal place moves to the left
Question 19
o The decimal place moves to the right
o The decimal place doesn't move at all Of a volumetric pipette mark with a ring near the mouth
end, the solution must be
Question 14
o Rinsed out
20°℃ is __ °F
o Blown out
o 68 o Drain out
o 53 o Left in the pipet before rinsing out with distilled
o 86 water
o 25
Question 20 Question 25

Reagent grade Type III water is used in All of the following describes positive displacement
micropipet except
o Highly sensitive procedures
o Rinsing or washing of glassware o With disposable piston
o Preparation of culture media o For volatile sample
o Preparation of buffer o Piston is a permanent part of the pipet
o No air cushion

Question 21

When using a micropipettor, if you push to the second

stop to fill it you will get

o Either too much or too little solution

o The correct amount of solution
o Too much solution
o Too little solution

Question 22

Which of these is a correct colligative property?

o Boiling point depression

o Density
o Vapor pressure lowering
o Freezing point elevation

Question 23

The boiling point of a solution is that of a pure solvent

o Higher than
o Equal to
o Lower than
o Less than

Question 24

Borosilicate glass is commercially known as

o Corning
o Pyrex
o Soft glass
o Soda lime glass
QUIZ 2A LEC Question 5

Question 1 Which condition is a common cause of stray light?

In absorption spectrophotometry A. Dispersion from second-order spectra

A. Percent transmittance is directly proportional to B. Improper wavelength calibration

concentration
C. Misaligned source lamp
B. Absorbance is directly proportional to concentration
D. Unstable source lamp voltage
C. Percent transmittance is directly proportional to the
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light path length
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dehydrates and atomizes a sample using

A. A thermoelectric semiconductor
Question 6
B. An electron gun
Which type of filter is best for measuring stray light?
C. A graphite capillary furnace
A. Sharp cutoff
D. A thermospray platform
B. Wratten

C. Neutral density
Question 3
D. Didymium
Which type of monochromator produces the purest
monochromatic light in the UV ra
Question 7
A. A prism and a variable exit slit
Which component is required in a spectrophotometer
B. A sharp cutoff filter and a variable exit slit
in order to produce a spectral absorbance curve?
C. Interference filters and a variable exit slit
A. Photodiode array
D. A diffraction grating and a fixed exit slit
B. Multiple monochromators
https://quizlet.com/202476638/clinical-chemistry-
C. Laser light source
instruments-mls-review-flash-cards/
D. A reference optical beam
Question 4
https://www.flashcardmachine.com/spectrophotometr
Which photodetector is most sensitive to low levels of
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C. Photodiode

D. Photomultiplier tube
Question 8 Question 12

Optical density is referred to as Optimally, where should your unknown concentrations

fall in your standard curve?
A. Absorbance
A. To the left of your lowest data point.
B. Transmittance
B. To the right of your highest data point.
C. Emission
C. Close the midpoint between your known
D. Percent
concentrations.

D. When possible, above the line of best fit.

Question 9
https://quizizz.com/admin/quiz/5e7e8cf1d05b8b001b5
According to the Beer-Lambert law, absorbance is f79a8/spectrophotometer

A. Inversely proportional to the concentration Question 13

B. Directly proportional to the log of the concentration A spectrophotometer can be used to

C. Directly proportional to the transmittance A. Determine the presence of a substance in a solution.

D. Directly proportional to the concentration of the B. Measure the concentration of an unknown solution.
solution
C. Identify an unknown substance

D. Determine the molecular composition of an unknown

Question 10 substance.

If the following reaction is known to obey Beer's law,

the X axis is the concentration of the solution and the Y
Question 14
axis is
Which of the following lamps provides a continuous
A. None of the options are correct
spectrum of radiant energy in the visible and near UV
B. Wavelength regions?

C. Peak weight A. Tungsten-filament

D. Percent transmittance B. Mercury vapor

C. Hydrogen

Question 11 D. Deuterium

Following the standard Beer's Law, given diluted volume

of 5, 10, 20 and 40 standard, what will be the
Question 15
appearance of the graph? (not sure huhu)
Which of the following isolates light within a narrow
A. A straight line on semilog paper
region of the spectrum?
B. A straight line on percent transmission
A. Monochromator
C. A curve line on semilog paper
B. Photovoltaic cell
D. A straight line on a linear paper
C. Photomultiplier tube

D. Detector
Question 16 D. micrometers (µm)

The reagent blank corrects for absorbance caused by

A. The color of reagents Question 20

B. Sample turbidity When performing spectrophotometer quality assurance

checks, what is the helmium oxide glass filter used to
C. All of these options are correct
assess?
D. Bilirubin and hemolysis
A. Wavelength accuracy
https://freezingblue.com/flashcards/327301/preview/c
B. Stray light
hem01-instrumentation-and-methods
C. Linearity

D. Absorbance accuracy
Question 17

A standard calibration curve is used in the laboratory for

Question 21
A. Calibration of reagents
Which of the following formulas is an expression of the
B. Maintenance of a satisfactory performance
Beer-Lambert law that is routinely applied to
C. Standardization of reagents spectrophotometric analysis?

D. Determination of the concentration of an unknown A. Asx Cs/Cu = Au

B. A 2-log%T

Question 18 C. Cux Cs/As = Au

Which of the following is not descriptive of a D. Aux Cs/As = Cu

photomultiplier tube?
Question 22

Which of the following may be associated with

A. Cannot be read with a chopper reflectance spectrophotometry as it relates to the dry
reagent slide technique?
B. Amplifies the initial signal received

C. Must be shielded from stray light

A. Reflectance values are linearly proportional to
D. Emits electrons proportionally to initial light transmission values
absorbed
B. Light projected to the slide at 180-degree angle
https://quizlet.com/155817851/ch-1-success-clinical-
chemistry-437-q-flash-cards/ C. Dye concentration directly proportional to
reflectance
Question 19
D. Unabsorbed, reflected light detected by
What units of measurement are traditionally applied to photodetector
radiant energy in the visible portion of the
electromagnetic spectrum? https://www.brainscape.com/flashcards/multiple-
choice-question-rt-3007910/packs/4841322?page=1
A. nanometers (nm)
https://quizlet.com/405594324/success-in-clinical-
B. millimeters (mm) laboratory-science-clinical-chemistry-instrumentation-
C. centimeters (cm) and-analytical-principles-1-35-flash-cards/
Question 23

Matching Type

Checked with standard absorbing solution or filters -

Linearity

Any wavelength outside of the band transmitted - Stray

light

Demonstrated when a change in concentration results –

Wavelength
QUIZ 1A LAB Question 7

Question 1 Pipets are used to measure and dispense small amounts

of liquids. You should draw the liquid into the pipet
If an acid is splashed on your skin, wash at once with
using your mouth.
A. plenty of water
False
B. soap
Question 8
C. weak base
Work areas should be kept clean and tidy.
D. oil
True
Question 2
Question 9
Ten percent sodium hydroxide is used for
All chemicals in the lab are to be considered dangerous.
A. removing grease
True
B. cleaning new pipettes
Question 10
C. routine washing
All work surfaces should be sanitized at the end of the
D. removing blood clots shift with a solution

Question 3 o 10% bleach

o Concentrated
You are allowed to enter the chemical o 5% bleach
preparation/storage area any time you need to get an o 5% phenol
item.
Question 11
False
Decontaminate hands after
Question 4
o contact with patient's skin
All unauthorized experiments are prohibited. o contact with blood or body fluids
A. True o all statements are correct
o removing gloves
B. False
Question 12
Question 5
When you finish working with chemicals, biological
Chipped or cracked glassware is okay to use specimens, and other lab substances, always
A. True o treat your hands with skin lotion
B. False o wipe your hands on a towel
o wipe your hands on your clothes
Question 6 o wash your hands thoroughly with soap and
water
Laboratory work can be started immediately upon
entering the laboratory even if the instructor is not yet Question 13
present.
If a piece of equipment is not working properly, stop,
A. True turn it off, and tell
B. False o the custodian
o your best friend in the class
o your lab partner
 o the instructor Question 19

Question 14 If a lab experiment is not completed, you should

Why are safety symbols important? o make up some results

o come in during lunch and finish while eating
o They tell you how to do an experiment
lunch
o They tell you specific dangers
o discuss the issue with your instructor
o So you know what to do
o sneak in after school and work alone
o They are cool
Question 20
Question 15
After completing an experiment, all chemical wastes
In a laboratory, the following should not be worn.
should be
o loose clothing
o taken home
o dangling jewelry
o disposed of according to your instructor's
o all items are correct
directions
o sandals
o dumped in the sink
Question 16 o left at your lab station for the next class

Long hair in the laboratory must be Question 21

o cut short If you do not understand a direction or part of a lab

o held away from the experiment with one hand procedure, you should
o always neatly groomed
o figure it out as you do the lab
o tied back or kept entirely out of the way with a
o ask the instructor before proceeding
hair band, hairpins, or other confining device
o skip it and go on to the next part
Question 17 o try several methods until something works

The term Standard Precautions refers to Question 22

o Treating only blood or blood-tinged specimens Approved eye protection devices (such as goggles) are
as infectious worn in the laboratory
o Assuming that every direct contact with a body
o any time chemicals, heat or glassware are used
fluid is not infectious
o To avoid eye strain
o All statements are correct
o to improve your vision
o Treating all specimens as if they are infectious
o only if you don't have corrective glasses
Question 18
Question 23
When gathering glassware and equipment for an
If a laboratory fire erupts, immediately
experiment, you should
o run for the fire extinguisher
o clean any glassware that appears dirty
o open the windows
o read all directions carefully to know what
o notify your instructor
equipment is necessary
o throw water on the fire
o examine all glassware to check for chips or
cracks Question 24
o All statements are correct
Flammable materials, like alcohol, should never be
dispensed or used near
 o another student QUIZ 2A LAB
o an open flame
Question 1
o an open door
o a sink In the proper use of a volumetric pipet calibrated to
deliver (TD), one should
Question 25
A. Drain the content but do not blow out
All infectious materials should be properly disposed in a
___ plastic bag and submitted to the lab technician for B. Blow out the solution immediately
proper disposal.
C. Wipe the outside of the pipet with cotton
o blue
o yellow D. Rinse the content several times to mix the solution
o green Question 2
o red
A piper with double ring at the mouth piece indicates
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,If%20a%20laboratory%20fire%20erupts%2C%20immed A. A blow out type of pipet
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g%20the%20lab. C. It is to be used only for non-viscous fluid

D. To be rinsed several times with the diluent and then

blow out

http://mt-lectures.blogspot.com/2011/11/introduction-
to-clinical-chemistry.html

Question 3

The following are quality control tips in pipetting except

A. Air must not be aspirated during pipeting

B. Use pipets that are clean, no chips and correct size

for the volume to be measure

C. Drain the pipet in a slightly slanting position

D. The tip of the pipet must be wiped with gauze or a

tissue after coming from the solution

Question 4

Device for rapid pipetting made of spring activated

plunger in a precision bore with disposable tip

o Serological pipet
o Dropper
o Micropipette
o Pasteur pipette
Question 5 Question 9

A pipet should be wiped off In spectrophotometry, which of the following is a

mathematical expression of the relationship between
o After lowering the meniscus to the calibration
absorbance and transmittance?
mark
o Never if it is a volumetric pipet o Au/Cu = As/Cs
o Before lowering the meniscus to the calibration o A = log %T
mark o QA-2-log %T
o Only if it is a TC (to contain) pipet o A=abc

https://www.studystack.com/flashcard-1429021 Question 10

Question 6 A solution that has a transmittance of 1.0% would have

an absorbance of
When using an automatic pipet calibrated TC (to
contain) o 99%
o 1.0
o It must be made horizontally when filling
o 2.0
o Depress the piston to the first stop and the
o 1%
second stop to empty
o Depress the piston to the second stop to fill and https://quizlet.com/255591129/harr-mls-review-
the first stop to empty chemistry-51-instrumentation-flash-cards/ ditto na
o Depress the piston to the second top to both fill lahat hayup
and empty

Question 7
Question 11
When using a TC pipet in getting blood sample, how will
A spectrophotometer is set at zero optical density by
you dispense the blood sample?
using a
o Slowly drain the sample and rinse the pipet
o Control
o This is used as a serological pipet
o Serum sample
o Blow out all the blood sample
o Blank
o Slowly drain the blood sample
o Standard
Question 8

To properly use a volumetric pipet calibrated "to

Question 12
deliver" (TD), one should (not sure)
Parts of a spectrophotometer are
o Rinse out the contents several times
o Drain the contents but do not blow out o Tungsten lamp, cuvet, prism, phototube
o Drain the contents to the lowest etched mark in o Tungsten lamp, slit, filter, cuvet,
the volumetric pipet phototube.galvanometer
o Wipe the outside following the delivery of the o Tungsten lamp, slit, grating or prism, slit, cuvet,
contents phototube, galvanometer
o Tungsten lamp, grating or prism, cuvet,
phototube, galvanometer
Question 13 Question 17

According to Beer's law, the absorbance is Which of the following components determines the
wavelength of light that will pass through the sample
o Inversely proportional to the concentration
cuvette in a spectrophotometer?
o Directly proportional to the concentration
o Inversely proportional to the square of the o Detector
concentration o Light source
o Proportional to the square of the concentration o Monochromator
o Potentiometer

Question 14
Question 18
Spectrophotometer isolate a narrow band pass by
means of Which type of photodetector has a linear array that
allows it to respond to a specific wavelength resulting in
o Filter
complete UV/visible spectrum analysis?
o Prisms and layer cells
o Prisms and filter o Photodiode array
o Prisms and grating o Phototube
o Photovoltaic cell
Question 15
o Photomultiplier tube
Which of the following lists represents the light path
Question 19
through the components of a spectrophotometer
beginning immediately after the light source? Which of the following pipets are classified as transfer
pipets?
o Monochromator, entrance slit, sample cuvet,
exit slit, detector o Micropipets
o Entrance slit, monochromator, exit slit, sample o Ostwald-Folin
cuvet, detector o Mohr
o Monochromator, sample cuvet, entrance slit, o Serological
detector, exit slit
Question 21
o Entrance slit, sample, cuvet. exit slit.
monochromator, detector Which of the following pipets is calibrated to the tip?
Question 16 o None of these pipets
o Serological
Which of the following blanks is used to compensate for
o Mohr
absorption of the color of the test sample before
o Both pipets
reagents are added?
Question 22
o Water bank
o Alcohol blank Matching Type
o Reagent blank
o Sample blank Pipette with a long cylindrical tube drawn out to a tip
and is calibrated for blow out and in uniform fractional
volume of measurement

Volumetric Pipette
Pipette calibrated for the total volume of liquid and
must be washed out completely or must be rinsed
thoroughly

To continue pipette

Pipette calibrated for blow out of any small amount of

liquid remaining in the tip after delivery

To deliver pipette

Pipette calibrated for one specified volume and used for

pipetting

Serological pipette
MT–CC 1.1 (Clinical Chemistry) LEC Basic scientific community with a uniform method of
Principles and Practices describing physical quantities. (to avoid confusion)
Clinical Chemistry 2. SI system units (SI) are based on the metric system
 Basic science that utilizes the specialty of 3. Several subclassifications exist within the SI
system, one of which is the basic unit
chemistry to study human beings in various
4. Basic units of measurement
stages of health and disease  Metre (meter) - length
 Second - time
 An applied science when analyses are  Ampere – electric current
performed on body fluids or tissue specimens  Candela – luminous intensity
to provide important information for the  Kilogram - mass
diagnosis and treatment of disease (provide  Mole – quantity of a substance
us kung healthy ba yung tao or meron siyang  Kelvin – dynamic temperature
disease) 5. Used most often
 Length
 The test must be performed accurately if the  Mass
 Volume – quantity of a substance in mole
results of lab analyses are to be useful to the
 A derived unit, as the name suggests, is a derivative or a
physician in diagnosing and treating patients mathematical function describing one of the basic units
o Accuracy – it represents how close your 1. Example: meters per second (m/s), used to
express velocity
measurements come to its true value. Some Non SI Units that have become acceptable
So dapat malapit na malapit yung
 Long-standing units – hour, minute, day, gram, liter
results mo sa true value. and plane angles expressed as degrees
o Precision – how close a series of 1. These units, although widely used, can’t
measurements of the same thing are to technically be categorized as either basic or
each other. Not necessarily na naging derived SI units.
very close unlike accuracy.  Standard prefixes when added to a given basic unit,
Measurements that are imprecise do not can indicate decimal fractions or multiple of that
unit
properly identify random errors, and 1. Example: 0.001 L can be expressed using the
that can yield a widespread result prefix milli, or 10-3. And since it requires
aiding now patient diagnosis and moving the decimal point three places to the
treatment. right, it can be written as 1 millimeter, or
abbreviated as 1mL.
 The use of high quality analytical methods 2. It may also be written in scientific notation
and instrumentation is essential to lab work as 1x10-3 L. Likewise, 1000 liters would use
the prefix of kilo (103) and could be written
 Concerns: as 1 kiloliter or expressed in scientific
notation as 1x103 L.
 Units of measure  When converting between prefixes, simply note the
 Basic laboratory supplies relationship between them based on whether you
 Introductory laboratory mathematics are changing to a smaller or larger prefix and the
 Specimen collection, processing and incremental factor between them
1. Example:
reporting
- If converting from one liter (1.0x100 or
Units of Measure 1) to milliliters (1.0x10-3 pr 0.001), the starting
 Laboratory result consists of two components: unit is larger than the desired unit by a factor of
1. Number related to the actual value 1000 or 103.
2. Label identifying the units - This means that the decimal place would be
3. Example: In glucose determination, the result moved to the right of one (1) three places, so
obtained is 120mg/dL, 120 is the actual value 1.0 liter (L) equals 1000 milliliters (mL).
and mg/dL is the label/unit - When changing 1000 milliliter (mL) to 1 liter (L),
4. The unit defines the physical quantity of the process is reversed and decimal point would
dimension such as mass, length, time or volume be moved three places to the left to become 1L.
 Systeme International d’unites (SI) – adopted  SI conversions
internationally in 1960, is preferred in clinical
laboratories and is the only system employed in
many countries.
1. Devised to provide to provide the global
 containment programs --- prepare reagents in-
house
Therefore, a thorough knowledge of chemicals,
standards, solutions, buffers and water requirements is
necessary.
 Reagent quality control records must be retained for 5
years.
 Reagents shall be used and controlled according to the
manufacturer’s recommendations.
 All reagents and chemicals, including solvents and
1. Convert to larger unit: move decimal point to materials used in tests and assays, should be of
left appropriate quality.
2. Convert to smaller unit: move decimal  Reagent should be purchased from reputable, approved
point to the right suppliers and should be accompanied by the certificate of
 The SI term for mass in kilogram, it is the only analysis, and the material safety data sheet.
basic unit that contains a prefix as part of its
naming convention. Chemicals
 Generally, the standard prefixes for mass use the  Analytic chemicals exist in varying grades of purity:
term gram rather than kilogram. 1. Analytic reagent (AR)
2. Ultrapure
3. Chemically pure CP)
4. United States Pharmacopeia (USP)
5. National Formulary (NF) and
technical or commercial grade
You should know when to use this chemicals with a grades
of purity
MOST COMMON GRADES
 ACS grade
1. A chemical grade of highest purity and meets or
exceeds purity standards set by American Chemical
Society
2. Acceptable for food, drug or medicinal use and can
be used for ACS application or for general
 Reporting of lab results is often expressed in procedures that require stringent quality
terms of substance concentration (e.g. moles) specification and a purity of ≥95%.
or the mass of a substance (e.g. mg/dL, g/dL,  Reagent grade
g/L, mmol/L and IU) rather than SI units. 1. High purity generally equal to ACS grade and
 It has been recommended that analytes be suitable for use in many laboratories and analytical
reported using moles of solute per volume of applications
solution (substance concentration) and the 2. Acceptable for food, drug or medicinal use
liter be used as the reference volume.  USP grade
1. A chemical grade of sufficient purity to meet or
Reagents exceed the requirements of USP
 Reagent – any substance producing a chemical 2. Acceptable for food, drug, or medicinal use; may be
reaction used for most laboratory purposes
 In a highly automated laboratory, there seems  NF grade
to be little need for reagent preparation. 1. A grade of sufficient purity to meet or exceed
 Reagents are ready-to-use form or in a kit form requirements of the National Formulary (NF)
(i.e. all necessary reagents and respective 2. The USP and the NF USP-NF jointly publish a book of
storage containers pre-package as a unit) public pharmacopeial standard for chemical and
requiring only the addition of water or buffer biological drug substances, dosage forms and
for reconstitution compounded preparations, medical devices and
dietary supplements
 Everything is prepared when we use the reagent
kit.  Lab grade
1. A chemical grade of relatively high quality with
 Periodically, especially in hospital laboratories
exact levels of impurities unknown
involved specialized analyses or method
2. Usually pure enough for educational applications
validation, one may still face preparing
3. While excellent for teaching and training, it is not
various reagents or solutions.
pure enough to be offered for food, drug or
 As a result of deterioration of reagents, supply
medicinal use of any kind
and demand or the institution of cost-
 Purified grade 2. TYPE 2: Purified water
1. Also called pure or practical grade and  Workhouse of the water world, employed for
indicates good quality chemicals meeting general laboratory use in things like culture
no official standard media preparation or buffer creation
2. Can be used in most cases for educational 3. TYPE 3: Primary grade water
applications  Water operating behind the scenes, used for non-
3. Not pure enough to be offered for food, critical work like rinsing beakers, filling water
drugs or medicinal use of any kind baths or feeding autoclaves
 Technical grade Solutions
1. Good quality chemical grade used for  Mixtures in which soluble particles are completely
commercial and industrial purposes dissolved in a liquid or gas
2. Not pure enough to be offered for food,  Made up of solute and solvent
drug or medicinal use of any kind

Reference Materials
 Primary standard is a highly-purified chemical
that can be measured directly to produce a
substance of exact known concentration and
purity.
 Secondary standard is a substance of lower
purity, with its concentration determined by
comparison with a primary standard.

Water Specifications
 Reagent grade water (RGW)
1. Suitable for reagent and standard preparation
2. Most procedures use distilled water or
deionized water
 Distilled water
1. Purified to remove almost all organic materials
2. Water may be distilled more than once and
 Solution Properties
each distillation cycle will remove
1. Concentration
impurities
 Percent solution
 Deionized water
3 expressions of percent solution
1. Produced from distilled water using either
o Weight per weight (w/w)
an anion or cation exchange resin followed
o Volume per volume (v/v)
by replacement of the removed particles
o Weight per volume (w/v) – use g/dL instead
with hydroxyl or hydrogen ions, respectively of percent
 Reverse osmosis – pumps water across a semi- o For v/v solutions, it is recommended that
permeable membrane and produces RO water gram per decilitre (g/dl) be used instead of
 Water can also be purified by ultrafiltration, UV percentage or % (v/v)
light, sterilization or ozone treatment
 Lab requirements generally call for reagent
grade water that, according to the Clinical and
Laboratory Standards Institute (CLSI), is
classified into needed for its one of six categories
based on the specifications use rather the
method of purification or preparation
 Categories
1. Clinical laboratory reagent
2. Special reagent water
3. Instrument feed water
4. Water supplied by method of manufacturer
5. Autoclave and wash water  Normality
o Least likely encountered; used in chemical
6. Commercial bottled purified water
titrations & chemical reagent classification
 Types o Defined as the number of gram equivalent
1. TYPE 1 : Ultrapure water weights per 1L of solution.
 Used for highly sensitive procedures
like HPLC, AAS and mammalian cell
culture
 o Valence – number of units that can
combine with 1 mole of hydrogen ions for
acids and hydroxyl ions for bases and the
number of electrons exchanged in
oxidation-reduction reactions
o The number of atoms/elements that can
combine for a particular compound;
therefore the equivalent weight is the -
Saturation
gram combining weight of a material Dilute and unsaturated – relatively little

o Normality is always equal to or greater solute or one which has been made to
than the molarity of that compounds lower solute concentration per volume of
o Normality was previously used for solvent as when making a dilution
reporting electrolyte values such as Na+,  Concentrated solution– large quantity of
K+, Cl- expressed as milliequivalent per solute in solution
liter (mEq/L), however, has been  Supersaturated – has an even greater
replaced with more familiar units of concentration of solute particles than a
millimoles/liter (mmol/L) saturated solution of the same substance
o Thermodynamically unstable - greater
 Molarity concentration of solute particles
o Number of moles per 1L of solution o Addition of a solute or mechanical agitation
o One mole of a substance equal its gram disturbs the supersaturated solution,
molecular weight (GMW), so the resulting in crystallization of any excess
customary units of molarity are material out of solution
moles/liter  Saturated – solution in which there is an
o The SI expression for concentration excess of undissolved solute particles
should be represented as mol/L, mmol/L,
µmol/L, nmol/L 2. Colligative properties
o Properties of solutions that depend on the
ratio of the number of solute particles to the
number of solvent molecules in a solution,
and not on the nature of the chemical species
present
o The number ratio can be related to the
various units for concentration of a solution,
 Molality
for example, molarity, molality, normality
(chemistry), etc.
o The assumption that solution properties are
independent of nature of solute particles of only
exact for ideal solutions and is approximate for
dilute real solutions.
o Colligative properties are a set of solution
properties that can be reasonably approximated
by assuming that the solution is ideal.
o Only properties which result from the dissolution
of nonvolatile solute in a volatile liquid solvent are
considered.
o They are essentially solvent properties which
are changed by the presence of the solute
o The solute particles displace some solvent
molecules in the liquid phase are therefore
reducing the concentration of the solvent, so
that colligative properties are independent of
the nature of the solute
o Affected by the
 Vapor pressure – pressure at which the
liquid solvent is in equilibrium with the
water vapor
o Vapor pressure lowering – the
 vapor pressure of a solvent in a
solution is always lower than
the vapor pressure of the pure
solvent
 Freezing point – temperature at
which the vapor pressures off the
solid phases are the same
o Freezing point depression – 4. Conductivity
the freezing points of solutions - A solution’s conductivity quality depends
are lower than that of the pure principally on the number of respective charges
solvent. It is directly of the ions present
proportional to the molality of - A measure of how well electricity passes through
the solute a solution
 Boiling point – temperature at which - expressed as ohms-1 or mho
the vapor pressure of the solvent Resistivity – reciprocal of conductivity
o Measure of a substance’s resistance to the
reaches one atmosphere
passage of electrical current
o Boiling point elevation – the o The primary application in the clinical
boiling point of solutions are laboratory is for assessing the purity of
higher than that of the pure water
solvent. It is directly
o Expressed as ohms
proportional to the molality of
5. Buffers
solute
- weak acids or bases and their related
 Osmotic pressure – the pressure salts that, as a result of their dissociation
opposing osmosis when a solvent flows characteristics, minimize changes in the
through a semipermeable membrane to hydrogen ion concentration
establish equilibrium between - Hydrogen ion concentration
compartments of differing is often expressed as pH
concentration - pH represents the negative or inverse log
 For a given solute-solvent mass ratio, of the hydrogen ion concentration
all colligative properties are inversely
proportional to solute molar mass
 All of the properties are only colligative
in the dilute limit—at higher
concentrations, the freezing point
depression, boiling point elevation,
vapor pressure elevation or depression,
and osmotic pressure are all dependent - Unlike a strong acid or base, which
on the chemical nature of the solvent dissociates almost completely, the
and the solute dissociation constant for a weak acid or
3. Redox potential – measure of the ability of a base solution tends to be very small,
solution to accept or donate electrons meaning little dissociation occurs
Reducing agents – substances that donate
electrons (lose electrons, oxidized/increases
in oxidation number)
Oxidizing agents – substances that accept
electrons (gain electrons, reduced/decreases in
oxidation number)
 Photometry
- Instruments that measure electromagnetic radiation have
several concepts and components in common.
- Most frequently used: photometry or specifically
absorbance or reflectance spectrophotometry.
- Photometry employs color and color variation to
determine the concentration of various substances.
- A photometric components is employed in many of the
automated analyzers and any person during clinical lab
techniques should understand the principles of
photometry.
- Photometry is the measurement of the luminous
intensity of light, or the amount of luminous light falling
on a surface from a light source.
- Photometric instruments measure this intensity of light
without consideration of wavelength.
Ionic strength – important aspect of buffers, - Spectrophotometry is the measurements of the intensity
particularly in separation techniques of light at selected wavelengths.
 The ionic strength of a solution is a measure of - In absorbance spectrophotometry the concentration of
the concentration of ions in that solution an unknown sample is determined by measuring its
 Ionic compounds, when dissolved in water, absorption of light at a particular wavelength and
dissociate into ions. The total electrolyte comparing it with the absorption of light by known
concentration in solution will affect important standard solutions measured at the same time with the
properties such as the dissociation constant or same wavelength
the solubility of different salts - The intensity of color is directly proportional to the
 One of the main characteristics of a solution concentration of the substance present.
with dissolved ions is the ionic strength.
 Can be molar (mol/L solution) or molal (mol/kg Standard solution – it is a solution containing known
solvent) and to avoid confusion, the units should concentration of the same substance to be determined.
be stated explicitly

- The use of spectrophotometry, or colorimetry as a means

Instrumentation if quantitative measurement depends primarily on two
factors: the color itself and the intensity of the color.
- All about analytical techniques, methods, - Any substance to be measured by spectrophotometry
instruments used in cc. In clinical laboratory, there must be naturally colored o must be capable of being
is a continuous need for quantitative techniques of colored.
measurement. - An example of a substance that is colored to begin with
- Instrumentation has become miniaturized which is hemglobin. Sugar (glucose) that is not colored to
enable the development of point of care of device. begin with but is capable of being colored by certain
- Technologically, sophisticated, automated anlayzers reaction and reagents.
are still based on the traditional approaches and - When spectophotometry is used as a method for
technologies. quantitative measurement, the unknown colored
- Employed photometer in special photometry, ions substance is compared with a similar substance known
selective, electrodes electrophoresis, nephilometry. strength (a standard solution)
- Analytical techniques and instrumentation provides - In absorbance spectrophotometry the absorbance units
us foundation for all measurements made in modern or values for serveral different concentrations of a
clinical laboratory standard solution are determined by spectrophotometry
and are plotted on graph paper.
Analytical techniques and Instrumentations - The resulting graph is known as a standard calibration
 Four basic categories of most measurement curve or Beer’s law plot.
techniques - Unknown specimens can then be read in the
- Spectrometry – spectrophotometry, atomic spectrophotometer and by use of their absorbance
absorption, and mass spectrophotometry. values, their concentrations determined from the
- Luminiscence – fluorescence, chemiluminescence calibration curve.
and nephelometry.
- Eletroanalytical methods – electrophoresis,
potentiometry, and amperometry.
- Chromatography – gas, liquid and thin layer
techniques.
 depends on the wavelength that is not absorbed. When
light is not absorbed, it is transmitted.
- A colored solution has color because of its physical
properties which result in its absorbing certain
wavelengths and tansmitting others.
- When white light is passed through a solution, part of the
NATURE OF LIGHT light is absorbed and the remaining light is transmitted.
- Light is a type of radiant energy and it travels in the - Meron na a-absorb, meron din na t-transmit
form of waves. The distance between waves is the
wavelength of light. ABSORBANCE AND TRASNMITTANCE OF LIGHT: BEER’S LAW
- Light is used to describe radiant energy with - Measurements by spectrophotometry is based on the
wavelength visible to the human eye or with reaction between the substance to be measured and the
wavelength bordering those visible to the human reagent, chemical, used to produce color.
eye. - The amount of color produced in a reaction between the
- Electromagnetic radiation includes a spectrum of substance to be measured and the reagent depends on the
energy, from short wavelength, highly energetic concentration of the substance.
gamma rays and X-rays on the left side to - Therefore, the intensity of the color is proportional to the
wavelengths of radio frequencies on the right side concentration of the subsatnce.
of the spectrum. - Beer’s law states that the concentraion of a substance is
directly proportional to the amount of light absorbed or
inversely proportional to the logarithm if the
transmitted light.
- As the law states, the depth at which the color is
determined must be constant. The depth of the solution
is regulated by the cuvette or container used to hold it.

Beer’s law is the basis for use of photometry in

quantitative measurement.

Any increase in the concentration of the color producing

substance will increase the amount of color seen.

EXPRESSION OF LIGHT TRASNMITTED OR ABSORBED

- There are two common methods of expressing the amount
of light transmitted or light absorbed by the solution.
- Visible ligt passes between these frequencies, with (another term for absorbed light is optical density, OD)
the color violet at the 400 nm wavelenth and red at - The units used to express the readings obtained by the
the 700 nm wavelength. electronic measuring device are either absorbance (A)
- The human eye responds to radiant energy, or light, units or percent transmittance (%T).
with wavelenth between 380 and 750 nm - Absorbance is an expression of the amount of light
- A nanometer is 1 X 10-9m absorbed by a solution.
- With modern pgotometric appaatues, shorter - Absorbance values are directly proportional to the
(ultraviolet) or longer (infrared) wavelenths can be concentration of the solution and can be plotted on linear
measured. graph paper to give a straight line.
- Modern instruments isolate a narrow wavelenth
range of the spectrum for measurements.
- Most instruments use filters (photometers) or prisms
or gratings (spectrometers) to select or isolate a
narrow range of the incident wavelength.
- The wavelength of light determines the color of the
light seen by the human eye. Every eye color that is - Percent transmittance is the amount of light that passes
seen is light of a particular wavelength. through a colored solution compared with the amount of
- A combination, or mixture of light evergy of different light that passes through a blank or standard solution.
wavelength is known as daylight or white light. When - The blank solution contains all the reagents used in the
light is passed through a filter, a prism, or a procedure, but it does not contain the unknown substance
diffraction grating, it can be broken into a spectrum being measured.
of visible color ranging from violet to red. - Blank Solution – to correct the color distribution of the
- The color of light seen in the visible spectrum reagent.
- Percent transmittance varies from 0 to 100 (%T), with
 equal divisions on viewing scale.

TYPES OF GRAPH PAPER

- As the concentration of the colored solution - The concentrations of the standard solutions are plotted
increases, the amount of light absorbed increases, on the horizontal axis (x). The transmittance or
and the percentage of light transmitted decreases. absorbance readings from the photometer are plotted
- The transmitted light does not decrease in direct along the vertical axis (y).
proportion to the concentration or color intensity of
the solution being measured.
- Percent transmittance readings plotted against
concentration will not give a straight line on linear
graph paper.
- A logarithmic relationship exists between %T and
concentration, so when %T is plotted against
concentration, a semi-logarithmic graph paper is
used to obtain a straight line. - When percentages are plotted against concentrations on
the semi- logarithmic paper, the porportional relationship
is direct and the necessary straight line graph is obtained
when the individual standard points are connected.
- The criteria on good standard curve
o The line is straight
o The line connects all points
o The line goes through the origin, or intersect of
the two axes.
- The origin of the graph paper is the point on the vertical
and horizontal axes where there is %T and zero
concentration.

- Linear graph paper can also be used to plot absorbance

readings because absorbance of wavelengths of light is
directly proportional to the concentration of the colored
solution being read.
o The readings must first be converted to logaritmic
values plotted on the vertical axis.

PREPARATION AND USE OF A STANDARD CURVE

- In research laboratories or in the preparation of a
new or special procedure, it may be necessary to
prepare a standard curve manually.
- Familiarity with the principles of how a standard
curve is constructed and use. INSTRUMENTS USED IN SPECTROPHOTOMETRY
- Semi-logarithmic graph paper is used to plot %T - Spectrophotometry is the basis for many of the
readings form the photometer because a logarithmic instruments used in clinical chemistry.
relationship exists between percent and - The primary reasons for this are ease of measurement,
concentration. satisfactory, accuracy and precision, and the suitability
- Horizontal axis of the semi-logarithmic graph paper of spectrophotometric techniques to use in automated
is a linear scale and the vertical axis is a logarithmic instruments.
scale - In this section, spectrophotometers and colorimeters are
members of this class.
- Most of the instruments used in photometry have some
means of isolating a narrow wavelength, or range of the
color spectrum for measurements.
- Instruments using filters are referred to as filter
photometers, and those using prisms or diffraction
grating are called spectrophotometers or
photoelectric colorimeters.
- Photometers utilize an electron device to compare
the actual color intensities of the solutions
measured.
- Spectrophotometer is really 2 components in a
single case, a spectrophotometer, a device for
producing light of a specific wavelength, the
monochomator; and a photometer a device for Hold the spectrophotometers on the top only.
measuring light intensity. - Barrier-layer cell
o Least expensive
PARTS OF SPECTROPHOTOMETERS
o Composed of light-sensitive materials such as
- Light source: the light source supplies the
selenium on iron plate with transparent layer
radiant energy used to analyze the sample. The
of silver.
most common source of light for work in the
o When exposed ti light, electrons in the light
visible or near-infrared region is the
sensitive material are excited and are
incandescent tungsten or tungsten-iodine
released into the highly conductive silver.
lamp. Most emitted radiant energy is near-
o No need for power source.
infrared. The most common lamps for UV work
are deuterium-discharge & mercury oil lamps.
- Photoemissive/phototube
- The wavelength isolator: a system of isolating
o Has photosensitive material that gives off
a desired wavelength and excluding others is
electrons when light energy strikes it.
called a monochromator.
o Composed of positively charged anode and
- Some filters are very simple, composed of one
negatively charged cathode enclosed in a
or two pieces of colored glass. The filter must
glass.
transmit a color that a solution can absorb.
o The cathode emits electrons jump over to the
- One common instruments amploys a diffraction
positively charged anode where they are
grating with a special plate and slit to reduce
collected and return through an external
the spectrum to the desired wavelength. The
measurable amount.
grating consists of a highly polished surface with
o Required an external source of energy.
numerous lines that break up white light into
spectrum.
- Photomultiplier
- By moving the spectrum behind a slit (the light
o Emits electrons proportionally to initial light
source must be movable). Only one particular
absrobed, convert radiant ebergy to
portion of the spectrum is allowed to pass
electrical energy.
through the narrow slit. The paricular band of
o Must be shielded from stray light.
light, or wavelength transmitted through the slit
o Ampplified the initial signal received
is indicated on a viewing scale on the machine.
o Has a very rapid response time
o Most senisitive to low levels of light.
- Photodiode array
o A type of photodetector that has
photoiodides ina line or arrangement
allowing it to respons to a specific
wavelength resulting in complete UV/visible
- The cuvette holds the sample to be analyzed in
spectrum analysis.
the path of the energy.
o Produces a spectral absorbance curve.
- Cuvettes
o are made from plastic, glass or quartz.
- The amount of light transmitted by the solution in
These are acceptable in visible work.
the cuvette is measured by the photoelectric cell,
o There are inexpensive plastic cuvettes
that may be suitable for some UV work. producing electrons in proportion to the amount of
light hitting it. The electrons are passed on to a
o Analytical cell or sample cell.
galvanometer, where they are measured.
- The galvanometer records the amount of current
that it receives from the photoelectric cell on a
special viewing scale (read-out device) on the
 spectrophotometer. The results are reported in
terms of T% (absorbance).

Quality Control Tests

- Wavelength accuracy is ensured when the
wavelength indicated on the control dial is the
actal wavelength of light passed by the
monochromator. Instrumentation
- This is checked with standard absorbing solution
or filters with maximum absorbance of known Atomic Absorption Spectrophotometer
wavelength.  Principle – Ground-state atoms absorb light at defined
- Wavelength calibration can be tested using rare- wavelengths
earth glass filters (didymium), or holmium oxide - Used to measure concentration by detecting the
glass filters or a stable chromogen solution. absorption of electromagnetic radiation by atoms
- Photoelectric accuracy can be checked by rather than by molecules
reading standard solutions of potassium - Line spectrum refers to the wavelength at which an
dichromate or potassium nitrate. atom absorbs light, each metal exhibits a specific line
spectrum
- The sample is atomized in a flame where the atoms
- Stray light refers to any wave lengths outside
of the metal to be quantified are maintained at ground
tha band transmitted by the monochromator. state
The most common causes of stray light are: - Then a beam of light from a hollow-cathode lamp
(HCL) is passed through a chopper to the flame
1. Reflections of light from stcratches on - The ground state atoms in the flame absorb the same
optical surfaces or form dust particles wavelengths of light from the HCL as the atoms emit
anywhere in the light path. when excited
2. Higher-order spectra produced by diffraction - The light not absorbed by the atoms is measured as a
gratings decrease in light intensity by the detector
- Stray light is detected by using cutoff filters. - The detector (photomultiplier tube) will selectively
read the pulsed light from the chopper that passes
through the flame and will not detect
any light emitted by the excited atoms when they
return to ground state
- The difference in the amount of light leaving the
HCL, and the amount of light measured by the
detector is indirectly proportional to the
concentration of the metal analyte in the sample
(A is directly proportional to the %T)
 Components
- Hollow-cathode lamp (light source) – consists
of an evacuated gas-tight chamber containing an
anode, a cylindrical cathode and an inert gas, such
as helium or argon
- Chopper – used to break the steady light into
pulsating light. It is a rotating wheel placed
between the flame and the source
- Burner head for flame – sample cell
- Monochromator
- Detector
- Readout device
 Applied voltage causes ionization of the gas, and these
excited ions are attracted to the cathode, where they
collide with the metal coating on the cathode,
knocking off atoms and causing atomic electrons to
 become excited TURBIDIMETRY
 When the electrons of the metal atoms from the 1. Measures light blocked as a decrease in the light
cathode return to the ground state, the transmitted through the solution, dependent on particle
characteristic light energy of the metal is size and concentration.
emitted 2. Uses a spectrophotometer for measurement, and is
 Vaporized metal atoms from the sample can be limited by the photometric accuracy and sensitivity of the
found in the flame. The flame serves as the
instrument.
sample cuvet in this instrument and bring the
analyte to its ground state 3. The problems inherent in turbidimetry
 The light produced in the HCL passes through a A. variation in particle size of samples
chopper (to avoid errors caused by B. variation in particle size of standards
measurement of light emitted by the sample) C. rate of aggregation or settling of particles
and then to the flame, and the light is absorbed
by the metal in the sample. The light not NEPHELOMETRY
absorbed will be read by the photomultiplier 1. The measurement of light scattered by a particulate
tube solution.
 A flameless system employs a carbon rod 2. Generally, scattered light is measured at an angle to
(graphite furnace), tantalum, or platinum to the incident light when small particles are involved; for
hold the sample being analyzed. The atomized large molecules, forward light scatter can be measured.
sample then absorbs the light energy from HCL. 3. The amount of scatter is directly proportional to the
This technique is more sensitive than the flame
number and size of particles present in the solution.
method.
4. The sensitivity of nephelometry depends on the
 In assaying analyte with a single-beam atomic
absorption spectrophotometer, the instrument
absence of background scatter from scratched cuvets and
is actually measuring the intensity of the beam particulate matter in reagents.
from the hollow cathode lamp after it has MOLECULAR EMISSION SPECTROSCOPY
passed through the analyte-containing flame I. Types of luminiscence where excitation does
 It is accurate, precise and specific, one requires absorption of radiant energy.
disadvantage is the inability of the flame to A. Fluorescence is the emission of light by a
dissociate samples into free atoms substance that has absorbed light or other
 Another possible theorem is the ionization of electromagnetic radiation.
atoms following dissociation by the flame,
which can be decreased by reducing the flame It is a form of luminiscence, in most cases, the emitted
temperature light has a longer wavelength, and therefore lower energy
- Matrix interference, due to the enhancement of
than the absorbed radiation.
light absorption by atoms in organic solvents or
formation of solid droplets as the solvent
I. Fluorometry is defined as the measurement of the
evaporates in the flame, can be another source emitted fluorescence light.
of error
- This interference may be overcome by The excitation light is absorbed by the atoms of the
pretreatment of the sample by extraction analyte in solution, which causes the electrons to move
to higher energy orbitals.
Upon return to ground state, light is emitted from the
fluorescing analyte and that light passes through a
secondary filter.
The secondary filter and the detector are placed at a right
angle to the light source to prevent incident light from
being measured by the detector.
Whereas fluorometers use filters, spectro-fluorometers
use prisms or diffraction gratings as monochromators.
2. Advantages: Fluorometry is about 1000 times more
sensitive than absorption techniques and has increased
specificity because optimal wavelengths are chosen both
for absorption (excitation) and for monitoring emitted
fluorescence.
3. Limitation: Changes from the established protocol that
affect pH, temperature, and solvent quality; self-
absorption: quenching.
Fluorometers are designed so that the path of exciting
light is at a right angle to the path of the emitted The solute components move at different rates because
light and this design prevents excitation light from of solubility in the mobile phase and electrostatic forces
reaching the detector. of the stationary phase that retard solute movement.
Requires primary and secondary monochromator. These two phase work together to provide solute
Primary advantage of performing fluorometric over resolution and separation.
absorption spectrosopic methods of analysis is a. Solute will stay with the solvent front if solvent is too
increased specificity and increased sensitivity. polar for the solute.
b. Phosphorescence is the emission of light b. Solute will remain at origin if solvent is insufficiently
produced by certain substances after they absorb polar.
energy. Basic steps in performing TLC include
It is similar to fluorescence except that the * sample extraction using a liquid-liquid or column
time delay is longer (greater than 10−4 sec) between technique
absorption of radiant energy and release of energy as * concentration of the extracted sample;
photons of light. * sample application by spotting onto the silica gel plate
2. Types of luminiscence where excitation does not * development of the solute in the sample using stationary
require absorption of radiant energy and mobile phases
a. Chemiluminiscence is the process where the * solute detection using chromogenic sprays, UV light,
chemical energy of a reaction produces excited fluorescence, and heat and
atoms and upon electron return to ground state, * interpretation of chromatographic results utilizing Rf
photons of light are emitted. values of solutes in comparison to aqueous standards.
Advantage: Has excellent sensitivity and dynamic In TLC, the Rf is the distance the solute migrates
range divided by the distance the solvent migrates.
Ruthenium is the substance used to generate light Rf values are affected by chamber saturation,
signal. temperature, humidity, and composition of the solvent.
Source lamp is not needed in chemiluminiscent GAS-LIQUID CHROMATOGRAPHY (GLC)
immunoassay, has monochromator, photodetector 1. Components include
and wash solution. * a carrier gas with a flow-control device to regulate the
b. Bioluminiscence is the process where an enzyme- gas flow
catalyzed chemical reaction produces light * a heated injector
emission. * chromatographic column to separate the solutes
For ex., this may occur in the presence of the enzyme * heated column oven
luciferase because of oxidation of the substrate * detector
luciferin. *computer to process the data and control the operation
Luminometer is a generic term for the type of of the system
instrument that is used to measure 2. Gas-liquid chromatography is a technique used to
chemiluminiscence and bioluminiscence. separate volatile solutes.
CHROMATOGRAPHY a. The sample is injected into the injector component of
1. A technique where solutes in a sample are the instrument where the sample is vaporized because
separated for identification based on physical the injector is maintained approximately 50°C higher
differences that allow their differential distribution than the column temperature.
between a mobile phase and a stationary phase. b. An inert carrier gas (mobile phase) carries the
2. Mobile phase may be an inert gas or liquid. vaporized sample into the column. Carrier gases
3. Stationary phase may be silica gel bound to the commonly used include hydrogen, helium, nitrogen and
surface of a glass plate or plastic sheet; may be silica argon.
or a polymer that is coated or bonded within a The carrier gas flow rate is critical to maintaining column
column. efficiency and reproducibility of elution times.
THIN-LAYER CHROMATOGRAPHY (TLC) The elution order of volatiles is usually based upon the
It is a type of planar chromatography. The stationary boiling point.
phase may be silica gel that is coated onto a solid c. The types of columns (stationary phase) used are
surface such as a glass plate or plastic sheet. designated as packed or capillary.
The mobile phase is a solvent, where solvent polarity When the volatile solutes carried by the gas over the
should be just enough to achieve clear separation of stationary phase of the column are eluted, the column
the solutes in the sample. It is used clinically for urine effluent is introduced to the detector.
drug screening. The solutes are introduced to the detector in the order
The mobile phase moves through the stationary that each was eluted.
phase by absorption and capillary action. d. The detector produces a signal for identification and
quantification of the solutes. A pre-column and guard column function to maintain the
Commonly used detectors include flame ionization, integrity of the column and are positioned prior to the
thermal conductivity, electron capture and mass sample reaching the main column.
spectrometer. The column, which functions as the stationary phase,
e. Separation of solutes is a function of the relative generally operates at room temperature.
differences between the vapor pressure of the The effluent from the column passes to a detector
solutes and the interactions of the solutes with the system.
stationary column. The solutes are introduced to the detector in the order
The more volatile a solute, the faster it will elute that each was eluted.
from the column; the less interaction of the solute 4.The detector produces a signal for identification and
with the column, the faster it will elute. quantification of the solutes.
f. Identification of a solute is based on its retention Commonly used detectors include spectrophotometer,
time, and quantification is based on peak size, photodiode array, fluorometer, electrochemical (glassy
where the amount of solute present is proportional carbon electrode)
to the size of the peak (area of height of the sample It separates solutes in a sample based on the sign and
peak, is compared to known standard). magnitude of the ionic charge.
In addition, GLC MASS SPECTROMETRY
a. separation depends on the sample and solubility in 1. A mass spectrometer is an instrument that uses the
the liquid layer of the stationary phase principle of charged particles moving through a magnetic
b. stationary phase is a liquid layer adsorbed on the or electric field with ions being separated from other
column packing. charged particles according to their mass-to-charge
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY ratios (parameter used to identify a compound)
(HPLC) In this system, electrons bombard a sample, ionizing the
1. HPLC components include compound into fragment ions, which are separated by
* sample-introduction system their mass-to-charge ratios.
* solvent reservoirs (solvent-delivery system) The mass spectrum produced is unique for a compound
* one or more pumps to propel the solvent(s) (identification), and the number of ions produced relates
* injector proportionally to concentration (quantification).
* chromatographic column 2. Mass spectrometry is a high-quality technique for
* detector identifying drugs or drug metabolites, amino acid
* computer to process data & control the operation composition of proteins, and steroids.
of the system In addition, mass spectrometry has applications in the
2. HPLC is a type of liquid chromatography where the field of proteomics.
mobile phase is a liquid that is passed over the The eluate gas from a gas chromatograph may be
stationary phase of the column. introduced into a mass spectrometer that functions as
The separation of solutes in a sample is governed by the detector system, or the liquid eluate may be
the selective distribution of the solutes between the introduced from a high-liquid chromatograph.
mobile and the stationary phase. 3. Instrumentation: Mass spectrometer components
a. Solvents commonly used for the mobile phase include
include acetonitrite, methanol, ethanol, isopropanol a. Ion source: samples enter the ion source and are
and water. bombarded by the ionization beam.
1. Isocratic elution: strength of solvent remains When the sample is in gas form and introduced from a gas
constant during separation chromatograph, the ion source may be electron or
2. Gradient elution: strength of solvent continually chemical ionization.
increases (%/min) during separation Other types, such as electrospray ionization and sonic
b. Stationary phase is an organic material covalently spray ionization, may be used when a high-performance
bonded to silica that may be polar or nonpolar in liquid chromatograph is used in conjunction with
composition. spectrometer.
1. Normal-phase liquid chromatography: polar b. Vacuum system: prevents the collision of ions with
stationary phase and nonpolar mobile phase other molecules (fragments) when electronic or magnetic
2. Reversed-phase liquid chromatography: separation is occurring.
nonpolar stationary and polar mobile phase c. Analyzer: Beam-type and trapping-type
3. The solvent-delivery system utilizes a solvent 1. Beam-type is a destructive process, where ions
reservoir from which the pump can push the mobile pass through the analyzer one time and then strike the
phase through the column. detector.
The sample is introduced through a loop injector. 2. Quadropole is a beam-type analyzer, where
mass-to charge ratios are scanned during a prescribed containing the analyte.
time period to form a mass spectrum. b. Current measured: voltage change versus plotted to
d. Detector usually detects ions using electron produce a polarogram. .
multipliers, such as discrete dynode and continuous c. Voltage at which sharp rise in current occurs
dynode electron multipliers. characteristic of the electrochemical reaction involved.
e. Computer and software convert the detector's d. Amount of increase in current (i.e. wave height)
signal to a digital form. proportional to the concentration of analyte.
Sample identification is achieved because
each compound produces a unique spectrum, which 2. Anodic stripping voltammetry is based on polarography
is analyzed by a database for matching to a a. Negative potential applied to one of the electrodes.
computerized reference library. b. Trace metal ions in the solution reduced and plated
4. To further improve selectivity and sensitivity, a onto anodic electrode; preconcentrating step.
system known as tandem mass spectrometers can be c. Plated electrode used as anode in polarographic cell;
employed, where a gas chromatograph is connected metal stripped off anode.
to two mass spectrometers (GC MS/MS) or d. Current flow during stripping provides polarogram that
(HPLC/MS/MS). identifies and quantifies the analyte being measured
In these systems, ions of a specific mass-to- (trace metals).
charge ratio are allowed to continue to the second e. Used to assay heavy metals such as lead in blood.
mass spectrometer where additional fragmentation
occurs and final analysis is done. 1. Potentiometry is a technique used to determine the
ELECTROCHEMISTRY concentration of a substance in solution employing an
1. When chemical energy is converted to an electrical electrochemical cell that consists of two half-cells,
current (a flow of electrons) in a galvanic cell, the where the potential difference between an indicator
term electrochemistry is used. electrode and a reference electrode is measured.
2. Electrochemical reactions are characterized by a a. Half cell, also called an electrode, composed of single
loss of electrons (oxidation) at the positive pole metallic conductor surrounded by solution of electrolyte.
(anode) and a simultaneous gain of electrons b. In an electrolytic cell, the half cell where reduction
(reduction) at the negative pole (cathode). takes place is a cathode.
3. The galvanic cell is made up of two parts called c. Two different half-cells connected to make complete
half-cells, each containing a metal in a solution of circuit; current flows because of potential difference
one of its salts. between two electrodes.
4. These methods involve the measurement of d. Salt bridge connection between two metallic
electrical signals associated with chemical systems conductors and between two electrolyte solutions.
that are within an electrochemical cell. e. Comparison made between the voltage of one half-cell
5. In the clinical lab, electroanalytical methods are connected to another half-cell.
used to measure ions, drugs, hormones, metals and f. Half-cell potentials compared to potential generated
gases. by standard electrode
6. Methods are available for the rapid analysis of g. Universally accepted standard half-cell is the standard
analytes present in relatively high concentrations in hydrogen electrode, arbitrally assigned a potential E° of
blood and urine, such as blood electrolytes (Na+, K+, 0.000 volt.
Cl-, HCO3-), and other analytes present in very low h. Desirable to use one half-cell (reference electrode)
concentrations, such as heavy metals and drug with known and constant potential, not sensitive to
metabolites. composition of material to be analyzed.
i. Calomel electrode type of reference electrode,
1. Many types of electrochemical analyses are used consisting of mercury covered by a layer of mercurous
including potentiometry, amperometry, chloride in contact with saturated solution of potassium
coulometry and polarography. chloride
2. The two basic electrochemical cells involved in j. Other half-cell (indicator electrode) selected on basis
these analyses are galvanic and electrolytic cells. of change in its potential with change in concentration of
Polarography, analytical procedure measuring analyte to be measured.
concentration of a substance based on the amount of k. Silver-silver chloride (Ag/AgCl) electrode; common
current flow resulting from ion transfer when type of reference electrode.
substance go into chemical reaction. 2. A pH/blood gas analyzer employs a pH-sensitive glass
It employs an electrochemical cell. electrode for measuring blood pH, and it employs PCO₂
a. Gradually increasing the voltage applied between and PO₂ electrodes for measuring gases in blood
two electrodes of the cell in contact with a solution For measuring pH, the pH electrode is a functioning glass
electrode that is dependent on properties of pH- ability to bind K+ used as a neutral carrier for K+ selective
sensitive glass membrane
When a pH-sensitive glass electrode is not actively d. NH4+ analysis: antibotics nonactin and monactin used
in use, it should be kept in a medium recommended in combination as neutral carrier for NH4+--selective
by the manufacturer membrane
To calibrate the pH electrode, it is necessary that 5. Sodium analysis: ion-selective electrodes based on
calibrating gases, two buffer solutions of known pH principle of potentiometry
be used a. Unlike glass membrane electrodes with selective
a. Glass electrode made by sealing thin piece of pH- capability
sensitive glass at the end of glass tubing and filling b. Constructed from glass that consists of silicon dioxide,
tube with solution of hydrochloric acid saturated with sodium oxide and aluminum oxide
silver chloride c. lon-selective electrode analysis of sodium
b. Silver wire immersed in tube's solution with one * uses a glass membrane
end extending outside the tube for external * error occur from protein buildup on the membrane
connection, silver-silver chloride reference electrode * principle is based on potentiometry
sealed within tube with pH-sensitive glass tip * membrane not coated with valinomycin
c. pH sensitive glass must be saturated with water. Amperometry: Electrochemical technique that measures
Surface of the glass develops a hydrated lattice, the amount of current (measures the increase in
allowing exchange of alkaline metal ions in the conductivity) produced through the oxidation or
lattice for hydrogen ions in the test solution. reduction of the substance to be measured at an
A potential is created between the inside and the electrode held at a fixed potential.
outside of the electrode, and that potential is 1. In a pH/blood analyzer, the electrode for
measured measuring the partial pressure of oxygen (PO₂) in
d. Glass electrode calibrated by comparison with two the blood is an electrochemical cell consisting of
primary standard buffers of known pH a platinum cathode and a Ag/AgCI anode
e. Because pH readings are temperature sensitive, connected to an external voltage source.
the calibration must be carried out at a constant 2. The cathode and anode are immersed in the
temperature of 37°C
buffer. A polyropylene membrane selectively
3. In a pH/blood gas analyzer, the PCO, electrode for
permeable to gases separates the electrodes and
measuring the partial pressure of carbon dioxide
(PCO₂) in blood is actually a pH electrode immersed buffer from the sample.
in a bicarbonate solution 3. When there is no oxygen diffusing into the buffer,
a. The bicarbonate solution is separated from the there is practically no current flowing between
sample by a membrane that is permeable in gaseous the cathode and the anode because they are
CO₂ but not to ionized substance such as H+ ions polarized.
b. When CO₂ from the sample diffuses across the 4. When oxygen diffuses into the buffer from a
membrane, it dissolves, forming carbonic acid and sample, it is reduced from the platinum cathode.
thus lowering the pH. 5. The electrons necessary for this reduction are
The measurement of CO₂ in blood by means of PCO2 produced at the anode. Hence, aa current flows;
electrode is dependent on the change in pH because the current is directly proportional to the PO2, in
of increased carbonic acid in the electrolyte the sample.
surrounding the electrodes.
The pH is inversely proportional to the log of the Coulometry
PCO₂. Hence the scale of the meter can be calibrated 1. A chloride coulometry employs a coulometric
directly in terms of PCO₂ system based on Faraday's law, which states that
4. The ion-exchange electrode is a type of in an electrochemical system, the number of
potentiometric, ion-selective electrode equivalent weight of a reactant oxidized or
a. The ion-selective electrode universally used is the
reduced is directly proportional to the quantity of
pH
electricity used in the reaction.
electrode
Consists of liquid ion-exchange membrane
made of inert solvent and ion-selective neutral The quantity of electricity is measured in coulombs.
carrier material Coulomb is the unit of electrical quantity; I
b. Collodion membrane may be used to separate coulomb of electricity flowing per minute
membrane solution from sample solution constitutes a current of I ampere.
c. K analysis: antibiotic valinomycin, because of its
 2. If the current is constant, the number of equal on a protein is the protein's isoelectric point.
equivalent weights of reactant oxidized or 6. Buffer solutions of pH 8.6 are generally used for serum
reduced depends only on the duration of the protein electrophoresis. Using agarose gel or cellulose
current. acetate at this alkaline pH, serum proteins take on a net
3. In the chloride coulometer, the negative charge and will migrate toward the anode (+).
electrochemical reaction is the generation of Albumin migrates the fastest toward the anode
and the gamma globulins remain closer to the cathode (-
Ag+ ions by the passage of a direct current
).
across a pair of silver electrodes immersed in
7.Visualizing the separated analyte: Following
a conducting solution containing the sample eletrophoresis, treat the support medium with
to be assayed for chloride. colorimetric stains or fluorescent chemicals.
Amido black B, Ponceau S and Coomassie brilliant blue
As the Ag+ ions are generated, they are stains are used for visualizing serum proteins.
immediately removed from solution by Silver nitrate is used for CSF proteins, fat red 7B and oil
combining with chloride to form insoluble red O are used for lipoproteins and nitrotetrazolium blue
silver chloride. is used for lactate dehydro-genase.
8. Detection and quantification of the separated protein
When all the chloride is precipitated; further is accomplished using densitometer.
generation of Ag+ ions causes an increase in 9. Commonly encountered problems in electrophoresis:
conductivity of the solution. a. Holes in staining pattern: Analyte present in too
4. The endpoint of the titration is indicated by high a concentration
the increase in conductivity of the solution. b. Very slow migration: Voltage too low
c. Sample precipitates in support: pH too high or
COULOMETRIC CHLORIDOMETEDS AND ANODIC low; excessive heat production
STRIPPING VOLTAMMETRY 10. Isoelectric focusing is a type of zone electrophoresis
Chloride ISEs have largely replaced coulometric in which protein separation is based on the isoelectric
titrations for the determination of chloride in body point (pl) of the proteins.
fluids. This method utilizes polyacrylamide or agarose gel
Anodic stripping voltametry was widely used for the containing a pH gradient formed by ampholytes in the
analysis of lead and is best measured by medium.
electrothermal (graphic furnace), atomic absorption When exposed to an electric field, the ampholytes
spectroscopy or, preferably ICP MS) migrate based on their pl to their respective positions in
ELECTROPHORESIS the gradient. In turn, the serum proteins will migrate in
1. Used clinically to separate and identify proteins, the gel to the position where the gel's pH equals the pl of
including serum, urine and cerebrospinal fluid (CSF) the respective protein.
proteins, lipoproteins, isoenzymes and so on. In turn, the serum proteins will migrate in the gel to the
2. Electrophoresis is defined as the movement of position where the gel's pH equals the pl of three
charged molecules in a medium when an electric field respective protein.
is applied. 11.Capillary electrophoresis is based on electroosmotic
3. Zone electrophoresis is defined as the movement flow (EOF). When an electric field is applied, the flow of
of charged molecules in a porous supporting medium liquid is in the direction of the cathode. Thus, EOF
where the molecules separate as distinct zones. regulates the speed at which solutes move through the
4. Support medium provides a matrix that allows capillary.
molecules to separate (e.g. agarose gel, starch gel, AUTOMATION PARAMETERS/TERMINOLOGY
polyacrylamide gel, and cellulose acetate 1. Centrifugal analysis: Centrifugal force moves samples
membranes. and reagents into cuvet areas for simultaneous analysis.
5. Movement of charged particles through a medium 2. Discrete analysis: Each sample reaction is
depends on the nature of the particle, including net compartmentalized.
charge, size and shape, the character of the buffer This may relate to an analyzer designed to assay
and supporting medium, temperature and the only one analyte (e.g. glucose) or an analyzer capable of
intensity of the electric field. forming multiple tests where the sample and reagents are
a. Nature of the charged particle. Proteins are in a separate cuvet/reaction vessel for each test.
amphoteric and may be charged positively or 3. Random access: Able to perform individual tests or
negatively depending on the pH of the buffer panels, and allows for stat samples to be added to run
solution. ahead of other specimens.
b. The pH at which negative and positive charges are 4. Batch analysis: Samples processed as a group.
5. Stand-alone: Instrument from a single discipline Reconstituting of reagents may need to be done
with automated capability. manually and then the reagents placed on an analyzer for
6. Automated-stand alone: Instrument from a single use, or reconstituting the reagents may be part of the
discipline with internal automated capability (e.g. total automation process as employed by Dimension®
auto-repeat and auto-dilute.) analyzer.
7. Modular workcell: At least two instruments from a b. Dry reagents can be spread over a support material and
single discipline with additional internal automated assembled into a single-use slide. This technique is
capability. employed by the Vitros® analyzer.
8. Multiple platform: Instrument able to perform c. Liquid reagents are pipetted by the instrument and
tests from at least two disciplines. mixed with the sample.
9. Integrated modular system: At least two analytical 4. Testing Phase
modules supported by one sample and reagent a. Mixing sample and reagents occurs in a vessel called a
processing and delivery system. cuvet. Some instruments have permanent, nondisposable
10. Pneumatic tube system: Transports specimens cuvets made of quartz glass. Other cuvets are made of
quickly from one location to another. plastic and are disposable.
11. Throughput: Maximum number of tests generated b. Reaction temperatures and times vary for each
per hour analyte. The most common reaction temperatures are
12.Turnaround: Amount of time to generate one 37°C and 30°C.
result c. Kinetic assays: Determination of sample concentration
13. Bar coding: mechanism for patient/sample is based on change in absorbance over time.
identification; used for regent identification by an d. Endpoint/colorimetric assays: Incubated for a specific
instrument. time, absorbance determined, absorbance related to
14. Dead volume: Amount of serum that cannot be calibrators for calculation of sample concentration.
aspirated e. A spectrophotometer is built within the system to read
15. Carry-over: The contamination of a sample by a absorbances for kinetic and colorimetric assays.
previously aspirated sample These systems may use a diffraction grating or aa
16. Reflex testing: Use of preliminary test results to series of high quality fibers. Some automated analyzers
determine if additional tests should be ordered or incorporate fluorometry or nephelometry.
cancelled on a particular specimen; performed 5. Data Management
manually or automated a. The computer module of most automated instruments
17. Total laboratory automation: Automated systems has a data management system that allows analysis of
exist for laboratories where samples are received, quality control (QC) materials and assessment of patient
centrifuged, distributed to particular instruments values (e.g. delta check) before releasing patient results.
using a conveyor system, and loaded into the b. Instruments/laboratory information systems (LISs) also
analyzer without operator assistance. archive patient results and QC values. These archived
This kind of automation is seen in large results are stored by the laboratory for various lengths of
medical center laboratories and commercial time.
laboratories where the volume of testing is high. POINT-OF-CARE TESTING (POCT)
1. Definition: Performing diagnostic tests outside the
main laboratory and at or near patient care areas.
PRINCIPLES OF AUTOMATION 2. Applications: POCT is designed to provide immediate
1. Automated instruments use robotics and fluidics to laboratory test results for immediate patient assessment
replicate manual tasks. and determination of appropriate treatment.
2. Specimen handling: Some instruments have level- POCT may be used in neonatal intensive care,
sensing probes that detect the amount of serum or coronary care, intensive care or the emergency
plasma in the tube. department.
Some systems have a reading device that 3. Operators
allows bar-coded sample tubes to be loaded onto the Only waived laboratory tests can be performed
instrument. using point-of-care instruments.
Although not as common, other instruments Clinical laboratory technicians and clinical
require the operator to manually enter the position laboratory scientists must operate instruments that
of the patient sample. perform complex or high-complexity laboratory tests.
3. Reagents 4. Point-of-care (POC) instrument evaluation
a. Dry reagents can be packaged as lyophilized All POC instruments must be evaluated in accordance
powder or tablet form that must be reconstituted with the Clinical Laboratory Improvement Amendments of
with a buffer or reagent grade water. 1988 (CLIA '88).
The values obtained from POC instruments must
correlate with values obtained from larger laboratory
instruments.
Linearity testing, calculation of control ranges,
correlations of sample data and reference ranges
must be done for each instrument
5. Training: All POC instrument operators must be
trained, and training must be documented.
6. Quality control: All effective quality control
systems must be set up for each POC instrument. The
program must use appropriate standards and
controls, statistical analyses, and a proficiency
testing system.
This information must be documented.
LONG QUIZ CC 2021 o Deionization
o Filtration
1. Glassware of choice for dilution and o Reverse osmosis
preparation of solutions that are made to o Distillation
definite volume 8. Which of these is a correct colligative property?
o Erlenmeyer Flask o Boiling point depression
o Volumetric Flask o Density
o Graduated Cylinder o Freezing point elevation
o Automatic pipet o Vapor pressure lowering
9. Mechanism that draws up and dispenses the liquid
2. All of the ff. describes positive displacement
is an integral part of the pipet
micropipette except
o Mohr pipet
o No air cushion
o Volumetric pipet
o With disposable piston
o Automatic pipet
o Piston is permanent part of the
o Serological pipet
pipet
10. Refers to how close a group of measurements are
o For volatile sample
to each other
o Estimate
3. The boiling point of a solution is ______ that
o Precision
of a pure solvent.
o Accuracy
o Lower than
o Significant
o Less than
11. Which of the ff. is material of known composition
o Higher than
available in a highly purified form?
o Equal to
o Control
4. The freeing point of a solution is _____ that
o Technical reagent
of a pure solvent.
o Test analyte
o Higher than
o Standard
o More than
12. When using a micropipette, if you push to the
o Equal to
second stop to fill it you will get
o Lower than
o The correct amount of solution
5. Which of the following terms refer to how
o The little solution
close an analytical value approaches the
o Too much solution
actual value of the substance during the
o Either too much or too little solution
assay?
13. Colligative properties of solutions are those
o Accuracy
properties that depend on
o Reliability
o The number of solute only
o Specificity
o Neither the number and nature of the
o Precision
solute
6. This is used for substances that are
o The nature of solute only
particularly sensitive to light such as
o Both the number and nature of solute
bilirubin.
14. Molality is
o Soft glass
o The moles of solute in 1g of solvent
o Low actinic glass
o The moles of solute in 1g of solution
o Borosilicate glass
o The moles of solute in 1 kg of solvent
o Soda-lime glass
o The moles of solute in 1kg of solution
https://labpedia.net/laboratory-glassware-cleaning-and- 15. What happens to a decimal place in the number
sterilization/ when you are converting from a large unit to a
smaller unit?
7. A method of purifying water where it is made o Converting doesn’t require a placement of
to pass over an insoluble resin polymer that a decimal point
exchange the hydrogen ions for the ionized o The decimal place moves to the left
impurities in the water. The principle is o The decimal place doesn’t move at all
 o The decimal moves to the right o Left in the pipet before rinsing out with
16. The chemical reagent has the highest purity distilled water
for laboratory use 24. Which of the ff. does not characterize borosilicate
o USP glass?
o None of the option is correct o Has a high thermal resistance
o Technical grade o Does not contaminate its content
o Analytical grade o Imparts amber/brown color to the glass
17. Reagent grade Type I water is used in o Has a low alkali content
o Highly sensitive procedures 25. Substance that resemble the unknown specimen
o Preparation of standards and used to measure precision
o All options are correct o Callibrator
o Procedures requiring minimal o Buffer
interference o Blank
18. Pipette with a long, cylindrical tube drawn o Control
out to a tip and is calibrated for blown out 26. Following the standard Beer’s Law, given diluted
and uniform fractional volume of volume of 5,10 ,20, and 40 standard what will be
measurement is known as the appearance of the graph? (di ko pa ren to
o To deliver pipette sure)
o Volumetric pipette o A straight line on percent transmission
o To contain pipette o A curve line on semilog paper
o Serological pipette o A straight line on semilog paper
19. The molecular weight divided by the valence o A straight line on a linear paper
is the 27. In absorption spectrometry
o Formula weight (mali yung asa quiz, tama na ito)
o Equivalent weight o Absorbance is directly proportional to
o Standard weight transmittance
o Molal weight o Percent transmittance is directly
20. Best glassware for preparation of reagent proportional to concentration
such as 100 mL of 10% sodium tungstate o Percent transmittance is directly
o 100 mL graduated cylinder proportional to the light path length
o 100 mL beaker o Absorbance is directly proportional to
o 100 mL Erlenmeyer flask concentration
o 100 mL volumetric flask 28. If the ff. reaction is known to obey Beer’s Law,
21. A molar solution contains the X axis is the concentration of the solution and
o None of the options is correct the Y axis is
o 1g equivalent weight of solute per o Absorbance
liter of solution o Percent transmittance
o 1g solute to 99g of solvent o Peak weight
o 1g molecular weight (gr.w) of solute o Wavelength
per liter of solution 29. Which measurement requires a primary and
22. An expression of one amount relative to secondary monochromator?
another o Spectrophotometer
o Molarity o Fluorometer
o Ratio o Atomic absorption spectrophotometer
o Dilution o Nephelometer
o Concentration 30. Similar to optical density
23. Of a volumetric pipette mark with a ring near o Emission
the end, the solution must be o Transmittance
o Drain out o Percent
o Rinsed out o Absorbance
o Blown out 31. Which substance is used to generate the light
signal in electrochiluminescence?
 o Ruthenium o Flow rate of the mobile phase is regulated
o Dioxetane phosphate o Mobile phase consists of constant solvent
o Luminol composition
o Acridinium o Stationary phase is equilibrated with the
32. Beer’s law states that the concentration of a mobile phase
substance is o Mobile phase is at constant temperature
o Concentration are all the same 39. Which type of filter is best for measuring stray
o Directly proportional to the square light?
root of a substance o Written
o Immersely proportional to the amount o Neutral density
of concentration o Didymium
o Directly proportional to the amount o Sharp cutoff
of the light absorbed 40. Which of the ff. is most commonly used detector
33. Reagent grade type III water is used in for clinical gas-liquid chromatography?
o Qualitative test in urinalysis o Flame ionization
o Both statements are correct o Both items are correct
o Rinsing or washing of glassware o Thermal conductance
o None of the statements are correct o Neither of these items are correct
34. A flameless atomic absorption 41. Which condition is a common cause of stray light?
spectrophotomter dehydrates and atomizes a o Dispersion from second-order spectra
sample using o Improper wavelength calibration
o An electon gun o Unstable source lamp voltage
o A thermospray platform o Misaligned source lamp
o A thermoelectric semiconductor 42. In thin-layer chromatography, the distance the
o A graphite capillary furnace solute migrates divided by the distance the
35. Chromatography is a physical method that is solvent migrates is the
used to separate and analyze o pK
o Viscous mixtures o tR
o Complex mixtures o Rf
o Metals o Kd
o Simple mixtures 43. Which photodetector is most sensitive to low
36. The term reverse phase is used in HPLC to levels of light?
indicate that the mobile phase is o Photomultiplier tube
o More polar than the stationary phase o Photodiode
o A stronger solvent than the stationary o Barrier layer cell
phase o Diode array
o Liquid and the stationary phase is 44. Why is vacuum system necessary in the mass filter
solid of a mass spectrophotometer?
o Organic and the stationary phase is o It prevents collision between
aqueous molecules/ions
37. Which component is required in a o Ionization doesn’t occur at atmospheric
spectrophotometer in order to produce a pressure
spectral absorbance curve? o It remove electrons form the ion source
o Multiple monochromators o It prevents contamination
o Laser light source
o Photodiode array
o A reference optical beam 45. Which type of monochromator produces the
purest monochromatic light in the UV range?
o Interference filter and a variable exit slit
38. The term isocratic is used in HPLC to mean o A prism and a variable exit slit
the o A sharp cutoff filter and a variable exit slit
o A diffraction grading and a fixed exit slit
46. In gas chromatography, the elution order of o The gas phase
volatiles is usually based upon the o The stationary phase
o Carbon content o The liquid phase
o Molecular size 54. Which of the ff. is not a problem inherent in
o Boiling point turbidimetry?
o Polarity o Need to maintain a constant and specific
47. Which of the following components is not temperature
needed in a chemiluminiscent immunoassay o Variation in particle size of samples
analyzer? o Variation in particle size of standards
o Photodetector o Rate of aggregation or settling of particles
o Source lamp 55. Fluorometers are designed so that the path of the
o Monochromator exciting light is at a right angle to the path of the
o Wash station emitted light. What is the purpose of this design?
48. Most commonly used solvents for the mobile o Prevent loss of the excitation light
phase in most HPLC is o Percent excitation light from reaching
o Acteonitrite the detector
o Hexane o Prevent loss of emitted light
o Chloroform o Focus emitted and excitation light upon
o Ethyl acetate the detector
49. Setting the spectrophotometer to zero is 56. Which of the ff represents a primary and
done using advantage of performing fluorometric over
o Water blank absorption spectroscopic methods of analysis?
o Reagent blank Ease of performing assays
o Sample blank Increased specificity and decreased sensitivity
o Any of the options is correct Increased specificity and increased sensitivity
50. Checked with standard absorbing solution or Purity of reagents used not as critical
filters 57. Which of the ff. may be associated with
o Stray light bioluminescence?
o Linearity o Electron excitation caused by radiant
o Wavelength energy
o All options are correct o Employs a radioactive label
51. What electrochemical technique measures o Less sensitive than direct fluorescent
the current produced through oxidation or assays
reduction between the indicator and o Light emission produced due to
reference electrodes held at a constant enzymatic oxidation of a substrate
electrical potential (voltage) in an 58. Which of the ff. best describes
electrochemical cell? chemiluminiscence?
o Amperometry o Electron excitation caused by radiant
o Coulometry energy
o Potentiometry o Enzymatic oxidation of a substrate
o Conductometry produces light emission
o Employs a radioactive label
o Chemical energy excites electrons that
emit light upon return to ground state.
52. What physical property is used to separate
59. Nephelometry is based on the measurement of
complex mixtures in chromatography?
light that is
o Density
o Absorbed by particles in suspension
o Hydrogen ion concentration
o Produced by excitation of ground state
o Vapor pressure
atoms
o Differential solubility
o Produced by fluorescence
53. The solvent is what part of a thin layer of
o Scattered by particles in suspension
chromatography system
o The mobile phase
60. In potentiometry, which of the ff. is
considered the standard electrode?
o Hydrogen electrode
o Calcium electrode
o Copper electrode
o Potassium electrode

Nephelometry
• The measurement of light scattered by a particulate
solution (big particles)
• Generally, scattered light is measured at an angle to
the incident light when small particles are involved;
for large molecules, forward light scatter can be
measured
• The amount of scatter is directly proportional to
the number and size of particles present in the
solution
• The sensitivity of nephelometry depends on the
absence of background scatter from scratched
cuvets and particulate matter in reagents

Turbidimetry
• Measures light blocked as a decrease in the light
transmitted through the solution, dependent on
particle size and concentration
• Uses a spectrophotometer for measurement, and
is limited by photometric accuracy and sensitivity of
the instrument
• The problems inherent in turbidimetry
- Variation in particle size of samples
- Variation in particle size of standards
- Rate of aggregation or settling of particles
Molecular Emission Spectroscopy radiant energy as photons of light

• Type of luminescence where excitation requires • Types of luminescence where excitation does not
absorption of radiant energy require absorption of radiant energy
- Fluorescence – emission of light by a substance - Chemoluminescence – chemical energy of a
that has absorbed light or other electromagnetic reaction produces excited atoms and upon
radiation electron return to ground state, photons of
➢ Emitted light has a longer wavelength, and light are emitted.
therefore, lower energy than the absorbed ➢ Advantage – has excellent sensitivity and
radiation dynamic range
➢ Fluorometry – measurement of the ➢ Ruthenium is the substance used to
emitted fluorescence light generate light signal
➢ The excitation light is absorbed by the ➢ Source lamp is not needed in
atoms of the analyte in solution, which chemiluminescent immunoassay, has
causes the electrons to move to higher monochromator, photodetector and wash
energy orbitals solution
➢ Upon return to ground state, light is - Bioluminescence – enzyme-catalyzed chemical
emitted from the fluorescing analyte and reaction produces light emission
that light passes through a secondary filter. ➢ This may occur in the presence of the
➢ The secondary filter and the detector are enzyme luciferase because of oxidation of
placed at a right angle to the light source to the substrate luciferin
prevent incident light from being ➢ Luminometer is a generic term for the type
measured by the detector of instrument that is used to measure
➢ Fluorometers use filters, chemiluminesce andbioluminescence
spectrofluorometers use prisms or
diffraction gratings as monochromators
➢ Advantage – fluorometry is about 1000x Chromatography
more sensitive than absorption techniques • Technique where solutes in a sample are separated
and has increased specificity because for identification based on physical differences
optimal wavelengths are chosen both for that allow their differential distribution between a
absorption (excitation) and for monitoring mobile phase and a stationary phase
emitted fluorescence • Mobile phase may be an inert gas or liquid
➢ Limitations – changes from the established • Stationary phase may be silica gel bound to the
protocol that affect pH, temperature, and surface of a glass plate or plastic sheet; may be
solvent quality; self-absorption; quenching silica or a polymer that is coated or bonded within a
column
➢ Fluorometers are designed so that the
Thin Layer Chromatography
path of exciting light is at a right angle
- Type of planar chromatography
to the path of the emitted light and
- The stationary phase may be silica gel that is
this design prevents excitation light
coated onto a solid surface such as a glass plate
from reaching the detector
or plastic sheet
➢ Requires primary and secondary
- The mobile phase is a solvent, where solvent
monochromator
polarity should be just enough to achieve clear
➢ Primary advantage of performing
separation of the solutes in the sample
fluorometric over absorption
- Used clinically for urine drug screening
spectrometric methods of analysis is - The mobile phase moves through the
increased specificity and increased stationary phase by absorption and capillary
sensitivity action
- Phosphorescence – emission of light
- The solute components move at different rates
produced by certain substances after
because of solubility in the mobile phase that
theyabsorb energy
retard solute movement
➢ It is similar to fluorescence except
that the time delay is longer (greater - These two phases work together to provide solute
than 10-4 sec) between absorption of resolution and separation
 ➢ Solute will stay with the solvent front if stationary phase
solvent is too polar for the solute - Solvents commonly used for the mobile phase
➢ Solute will remain at origin if solvent is include acetonitrite, methanol, ethanol,
insufficiently polar isopropanol and water
- Basic steps in performing TLC include ➢ Isocratic elution – strength of solvent
➢ Sample extraction using a liquid-liquid or remains constant during separation
column technique ➢ Gradient elution – strength of solvent
➢ Concentration of the extracted sample continually increases (%/min) during
➢ Sample application by spotting onto the separation
silica gel plate - Stationary phase is an organic material
➢ Development of the solute in the sample covalently bonded to silica that may be polar or
using stationary and mobile phases nonpolar in composition
➢ Solute detection using chromogenic ➢ Normal-phase liquid chromatography –
sprays, UV light, fluorescence, and heat polar stationary phase and nonpolar
➢ Interpretation of chromatographic results mobile phase
utilizing Rf values of solutes in comparison ➢ Reversed-phase liquid chromatography –
to aqueous standards nonpolar stationary and polar mobile phase
- In TLC, Rf is the distance the solute migrates - The solvent-delivery system utilizes a solvent
divided by the distance the solvent migrates reservoir from which the pump can punch the
- Rf values are affected by chamber saturation, mobile phase through the column
temperature, humidity, and composition of the ➢ The sample is introduced through a loop
solvent injector
➢ A pre-column and guard column function to
Gas-Liquid Chromatography maintain the integrity of the column and are
• Components include positioned prior to the sample reaching the
- A carrier gas with a flow-control device to main column
regulate the gas flow ➢ The column, which functions as the stationary
- A heated injected injector phase, generally operates at room temperature
- Chromatographic column to separate the solute ➢ The effluent from the column passes to a
- Heated column oven detector system
- Detector ➢ The solutes are introduced to the detector in the
- Computer to process the data and control the order that each was eluted
operation of the system - The detector produces a signal for identification and
• Gas-liquid chromatography is a technique used to quantification of the solutes
separate volatile solutes ➢ Commonly used detectors include
High-Performance Liquid Chromatography (HPLC) spectrophotometer, photodiode array,
• HPLC components include fluorometer, electrochemical (glassy carbon
- Sample-introduction system electrode)
- Solvent reservoirs (solvent-delivery) system ➢ It separates solutes in a sample based on the sign
- One or more pumps to propel the solvent(s) and magnitude of the ionic charge
- Injector
- Chromatographic column Mass Spectrometry
- Detector • A mass spectrometer is an instrument that uses the
- Computer to process data and control the principle of charged particles moving through a
operation of the system magnetic or electric field with ions being separated
• HPLC is a type of liquid chromatography from other charged particles according to their mass- to-
where the mobile phase is a liquid that is charge ratios (parameter used to identify a compound)
passed over the stationary phase of the - In this system, electrons bombard a sample, ionizing
column the compound into fragment ions, which are separated
- The separation of solutes in a sample is by their mass-to-charge ratios
governed by the selective distribution of - The mass spectrum produced is unique for a
the solutes between the mobile and the compound (identification), and the number of ions
produced relates proportionally to concentration
 (quantification) second mass spectrometer where additional
• Mass spectrometry is a high-quality technique for fragmentation occurs and final analysis is done
identifying drugs or drug metabolites, amino acid
composition of proteins, and steroids - The sample injected into the injector component of the
- In addition, mass spectrometry has applications in instrument where the sample is vaporized because the
the field of proteomics injector is maintained approximately 50°C higher than
- The eluate gas from a gas chromatograph may be the column temperature
introduced into a mass spectrometer that - An inert carrier gas (mobile phase) carries the
functions as the detector system, or the liquid vaporized sample into the column. Carrier gases
eluate may be introduced from a high-liquid commonly used include hydrogen, helium, nitrogen,
chromatograph and argon.
• Instrumentation – mass spectrometer ➢ The carrier gas flow rate is critical to maintaining
components include column efficiency and reproducibility of elution
- Ion source – samples enter the ion source and are times.
bombarded by the ionization beam ➢ The elution order of volatiles is usually based
➢ When the sample is in gas form and upon the boiling point
introduced from a gas chromatograph, the - The types of columns (stationary phase) used are
ion source may be electron or chemical designated as packed or capillary
ionization ➢ When the volatile solutes carried by the gas over
➢ Other types, such as electrospray the stationary phase of the column are eluted,
ionization and sonic spray ionization, may the column effluent is introduced to the
be used when a high-performance liquid detector
chromatograph is used in conjunction with ➢ The solutes are introduced to the detector in the
spectrometer order that each was eluted
- Vacuum system – prevents the collision of ions ➢The detector produces a signal for
with other molecules when electronic or magnetic identification and quantification of the
separation is occurring solutes Commonly used detectors include
- Analyzer – beam-type and trapping-type flame ionization, thermal conductivity,
➢ Beam-type is a destructive process, electron capture and mass spectrometer
where ions pass through the analyzer - Separator of solutes is a function of the relative
one time and then strike the detector differences between the vapor pressure of the
➢ Quadropole is a beam-type analyzer, solutes and the interactions of the solutes with the
where mass-to-charge ratios are stationary column
scanned during a prescribed time ➢ The more volatile a solute, the faster it will
period to form a mass spectrum elute from the column; the less interaction
- Detector usually detects ions using of the solute with the column, the faster it
electron multipliers, such as discrete will elute
dynode and continuous dynode electron - Identification of a solute is based on its
multipliers retention time, and quantification is based on
- Computer and software convert the peak size, where the amount of solute present is
detector’s signal to a digital form proportional to the size of the peak (area of
➢ Sample identification is achieved height of the sample peak, is compared to the
because each compound produces a known standard)
unique spectrum, which is analyzed by ➢ In addition, GLC…
a database for matching to a o Separation depends on the sample and
computerized reference library solubility in the liquid layer of the
• To further improve selectivity and sensitivity, stationary phase
a system known as tandem mass o Stationary phase is a liquid layer
spectrometers can be employed, where a gas absorbed on the column packing
chromatograph is connected to two mass
spectrometers (GC MS/MS) or (HPLC/MS/MS) Reflectance Spectrophotometry
- In these systems, ions of a specific mass-to- • Associated with unabsorbed, reflected light
charge ratio are allowed to continue to the detected by the photodetector as it relates to the dry
 reagent slide technique coulometry, and polarometry
• Measures the concentration of glucose in the • The two basic electrochemical cells involved in these
blood by using dry film technology analyses are galvanic and electrolytic cells
• Associated with plane-polarized light for • Polarography – analytical procedure measuring
sample excitation concentration of a substance based on the amount of
current flow resulting from ion transfer when
Fluorescence Polarization substance go into chemical reaction; employs an
• When the sample (fluorophore) is excited, it electrochemical cell
emits polarized light among the same plane as - Gradually increasing the voltage applied between two
the incident light if the fluorophore does not electrodes of the cell in contact with a solution
rotate in solution (i.e., it is attached or bound to containing the analyte
a large molecule) - Current measured – voltage change versus plotted to
• In contrast, a small molecule emits produce a polarogram
depolarized light because it will rotate out of - Voltage at which sharp rise in current occurs
the plane of polarization during its excitation characteristic of the electrochemical reaction involved
lifetime - Amount of increase in current (i.e., wave height)
• This technique is widely used for the proportional to the concentration of analyte
detection of therapeutic and abuseddrugs • Anodic voltammetry is based on polarography
• In the procedure, the sample analyte is - Negativepotentialappliedtooneoftheelectrodes
allowed to compete with a fluorophore- - Trace metal ions in the solution reduced and plated
labeled analyte for a limites antibody to onto anodic electrode;pre-concentrating step
analyte - Plated electrode used as anode in polarographic cell;
• The lower the concentration of the sample metal stripped off anode
analyte, the higher the macromolecular - Current flow during stripping provides polarogram
antibody-analyte- fluorophore formed and that identifies and quantifies the analyte being
lower the depolarization of the radiant light measured (trace metals)
- Used to assay heavy metals such as lead in blood
Electrochemistry • Potentiometry – technique used to determine the
• When chemical energy is converted into an concentration of a substance in solution employing an
electrical current (flow of electrons) in a electrochemical cell that consists of two half-cells,
galvanic cell, the term electrochemistry is used where the potential difference between an indicator
• Electrochemical reactions are characterized by electrode and a reference electrode is measured
a loss of electrons (oxidation) at the - Half-cell, also called an electrode, composed of single
positive pole (anode) and a simultaneous metallic conductor surrounded by solution of
gain of electrons (reduction) at the negative electrolyte
pole (cathode) - In an electrolytic cell, the half cell where reduction takes
• The galvanic cell is made up of two parts called half- place is a cathode
cells, each containing a metal in a solution of one of its - Two different half-cells connected to make complete
salts circuit; current flows because of potential difference
• These methods involve the measurement of between two electrodes
electrical signals associated with chemical systems - Salt bridge connection between two metallic
that are within an electrochemical cell conductors and between two electrolyte
• In the clinical lab, electroanalytical methods are used
solutions
to measure ions, drugs, hormones, metals and gases - Comparison made between the voltage of one
• Methods are available for the rapid analysis of
half-cell compared to another half-cell
analytes present in relatively high concentrationsin - Half-cell potentials compared to potential
blood and urine, such as blood electrolytes (Na+, K+, generated by standard electrode
Cl-
- Universally accepted standard half-cell is the
, HCO3-), and other analytes present in very low
standard hydrogen electrode, arbitrally
concentrations, such as heavy metals and drug
assigned a potential E0 of 0.000 volt
metabolites
- Desirable to use one half-cell (reference electrode)
• Many types of electrochemical analyses are used
with known and constant potential, not
including potentiometry, amperometry,
sensitive to composition of material to be
 analyzed of 37°C
- Calomel electrode – type of reference - In a pH/blood gas analyzer, the PCO2 electrode
electrode, consisting of mercury covered for measuring the partial pressure of carbon
by a layer of mercurous chloride in contact dioxide (PCO2) in blood is actually a pH electrode
with saturated solution of potassium immersed in a bicarbonate solution
chloride ➢ The bicarbonate solution is separated from
- Other half-cell (indicator electrode) the sample by a membrane that is
selected on basis of change in its potential permeable in gaseous CO2 but not to
with change in concentration of analyte to ionized substance such as H+ ions
be measured ➢ When CO2 from the sample diffuses across the
- Silver-silver chloride (Ag/AgCl) electrode; membrane,itdissolves,formingcarbonicacid and
common type of reference electrode thus lowering the pH
• A pH/blood gas analyzer employs a pH- o The measurement of CO2 in blood b means
sensitive glass electrode for measuring blood of PCO2 electrode is dependent on the
pH, and employs PCO2 and PO2 electrodes for change in pH because of increased carbonic
measuring gases in blood acid in the electrolyte surrounding the
- For measuring pH, the pH electrode is a electrodes
functioning glass electrode that is o The pH is inversely proportional to the log of
dependent on properties of pH-sensitive the PCO2. Hence, the scale of meter can be
glass calibrates directly in terms of PCO2
- When a pH-sensitive glass electrode is not - The ion-exchange electrode is a type of
actively in use, it should be kept in a potentiometric, ion-selective electrode
medium recommended by the ➢ The ion-selective electrode universally used is the
manufacturer pH electrode
- To calibrate the pH electrode, it is o Consists of liquid ion-exchange membrane
necessary that calibrating gases, two made of inert solvent and ion- selective
buffer solutions of known pH be used neutral carrier material
➢ Colloidon membrane may be used to separate
➢ Glass electrode made by sealing thin
piece of pH-sensitive glass at the end membrane solution from sample solution
➢ K+ analysis – antibiotic valinomycin, because of
of glass tubing and filling tube with
solution of HCl saturated with AgCl its ability to bind K+ used as a natural carrier for
➢ Silver wire immersed in tube’s
K+ selective membrane
solution with one end extending ➢ NH4+ analysis – antibiotics nonactin and
outside the tube for external monactin used in combination as neutral carrier
for NH4+-selective membrane
connection, silver-silver chloride
- Sodium analysis – ion-selective electrodes based
reference electrode sealed within on principle of potentiometry
tube with pH-sensitive glass tip ➢ Unlike glass membrane, electrodes with
➢ pH sensitive glass must be saturated
selective capability
with water. Surface of the glass ➢ Constructed from glass that consists of silicon
develops a hydrated lattice, allowing dioxide, sodium oxide and aluminum oxide
exchange of alkaline metal ions in the ➢ Ion-selective electrode analysis of sodium
lattice for hydrogen ions in the test o Uses a glass membrane
solution o Error occur from protein build-up on the
o A potential is created between the membrane
inside and the outside of the o Principle is based on potentiometry
electrode, and that potential is o Membrane not coated with valinomycin
measured • Amperometry – electrochemical technique that
➢ Glass electrode calibrated by measures the amount of current produced through the
comparison with two primary oxidation or reduction of the substance to be measured
standard buffers of known pH at an electrode held at fixed potential
➢ Because pH readings are temperature - In a pH/blood analyzer, the electrode for measuring
sensitive, the calibration must be the PO2 in the blood is an electrochemical cell
carried out at a constant temperature consisting of a platinum cathode and a Ag/AgCl anode
 connected to an external voltage source measured by electrothermal (graphic furnace),
- The cathode and anode are immersed in the atomic absorption spectroscopy or, preferably ICP-
buffer. A polyropylene membrane selectively M
permeable to gases separates the electrodes and
buffer from the sample Electrophoresis
- When there is no oxygen diffusing into the buffer, • Used clinically to separate and identify proteins,
there is practically no current flowing between the including serum, urine and CSF proteins,
cathodeand the anode because they are polarized lipoproteins, isoenzymes and so on
- When oxygen diffuses into the buffer from a • Movement of charged molecules in a medium when
sample, it is reduced from the platinum cathode an electric field is applied
- The electrons necessary for this reduction are • Zone electrophoresis is defined as the movement
produced at the anode. Hence, aa current flows; of charged molecules in a porous supporting
the current is directly proportional to the medium where the molecules separate as distinct
PO2 in the sample zones
• Coulometry • Support medium provides a matrix that allows
- A chloride coulometry employs a molecules to separate (e.g., agarose gel, starchgel,
soulometric system based on Faraday’s law, polyacrylamide gel and cellulose acetate
which states that in an electrochemical membranes)
system, the number of equivalent weight of • Movement of charged particles through a medium
a reactant oxidized or reduced is directly depends on the nature of the particle, including net
proportional to the quantity of electricity charge, size and shape, the character of the buffer
used in the reaction and supporting medium, temperature and the
- The quantity of electricity is measured in intensity of the electric field
coulombs. Coulomb is the unit of electrical - Nature of the charged particle – proteins are
quality. I coulomb of electricity flowing per amphoteric and may be charged positively or
minute constitutes a current of I ampere negatively depending on the pH of the buffer solution
- If the current is constant, the number of - The pH at which negative and positive charges are equal
equivalent weights of reactant oxidized or on a protein is a protein’s isoelectric point
reduced depends only on the duration of • Buffer solutions of pH 8.6 are generally used for serum
the current protein electrophoresis. Using agarose gel or cellulose
- In the chloride coulometer, the acetate at this alkaline pH, serum proteins take on a net
electrochemical reaction is the generation negative charge and will migrate toward the anode (+)
of Ag ions by the passage of a direct current - Albumin migrates the fastest toward the anode and
across a pair of silver electrodes immersed in the gamma globulins remain closer to the cathode (-)
a conducting solution containing the sample • Visualizing the separated analyte – following
to be assayed for chloride electrophoresis, treat the support medium with
➢ As the Ag+ are generated, they are colorimetric stains or fluorescent chemicals
immediately removed from solution - Amido black B, Ponceau S and Coomassie brilliant
by combining with chloride to form blue stains are used for visualizing serum proteins
insoluble AgCl - Silver nitrate is used for CSF proteins, fat red 7B and oil
➢ When all the chloride is precipitated; red O are used for lipoproteins and nitrotetrazolium
further generation of Ag+ causes an blue is used for lactate dehydrogenase
increase in conductivity of the solution • Detection and quantification of the separated protein is
- The endpoint of the titration is indicated by accomplished using densitometer
the increase in conductivity of the solution • Commonly encountered problems in electrophoresis
- Holes in staining pattern – analyte present in too high
Coulometric chloridometers and Anodic stripping concentrations
voltammetry - Very slow migration – voltage too low
• Chloride ISEs have largely replaced - Sample precipitates in support – pH too high or too
coulometric titrations for the determination low; excessive heat production
of chloride in body fluids • Isoelectric focusing is a type of zone electrophoresis in
• Anodic stripping voltammetry was widely which protein separation is based on the isoelectric
used for the analysis of lead and is best point (pI) of the proteins
 -This method utilizes polyacrilamide or agarose gel be aspirated
containing a pH gradient formed by ampholytes in • Carry-over – the contamination of a sample by a
the medium previously aspirated sample
- When exposed to an electric field, the ampholytes • Reflex testing – use of preliminary test results to
migrate based on their pI to their respective determine if additional tests should be ordered or
positions in the gradient. In turn, the serum cancelled on a particular specimen; performed
proteins will migrate in the gel to the position manually or automated
where the gel’s pH equals the pI of the respective • Total laboratory automation – automated systems
protein exist for laboratories where samples are received,
• Capillary electrophoresis is based on electrosonic centrifuged, distributed to particular instruments
flow (EOF) – when an electric field is applied, the flow using a conveyor system, and loaded into the
of liquid is in the direction of the cathode. Thus, EOF analyzer without operator assistance
regulates the speed at which solutes move through - This kind of automation is seen in large medical
the capillary center laboratories where the volume of testing
Automation Parameters/Terminology is high
• Centrifugal analysis – centrifugal force moves
samples and reagents into cuvet areas for
aimultaneous analsis Principles of Automation
• Discrete analysis – each sample reaction is • Automated instruments use robotics and fluidics
compartmentalized to replicate manual tasks
This may relate to an analyzer designed to • Specimen handling – some instruments have level-
assay only one analyte (e.g. glucose) or an sending probes that detect the amount of serum or
analyzer capable of forming multiple plasma in the tube
tests where the sample and reagents are - Some systems have a reading device that
in a separate cuvet/reaction vessel for allows bar-coded sample tubes to be loaded
each test onto the instrument
• Random access – able to perform individual - Although not as common, other instruments
tests or panels, and allows for stat samples to require the operator to manually enter the
be added to run ahead of other specimens position of the patient sample
• Batch analysis – samples processed as a group • Reagents
• Stand alone – instrument from a single disciple - Dry reagents can be packaged a lyophilized
with automated capability powder or tablet form that must be
• Automated stand alone – instrument from a reconstituted with a buffer or reagent-grade
single discipline with internal automated water
capability (e.g. auto-repeat and auto-dilute) - Reconstituting of reagents may need to be done
• Modular workcell – at least two instruments manually and then the reagents placed on an analyzer
from a single discipline with additional for use, or reconstituting the reagents as employed by
automated capability Dimension® analyzer
• Multiple platform – instrument able to - Dry reagents can be spread over a support material
perform tests from at least two disciplines and assemble into a single-use slide. This technique is
• Integrated modular system – at least two employed by Vitros® analyzer
analytical modules supported by one sample - Liquid reagents are pipette by the instrument and
and reagent processing and delivery system mixed with the sample
• Pneumatic tube system – transports • Testing Phase
specimens quickly from one location to - Mixing sample and reagents occurs in a vessel called a
another cuvet. Come instruments have permanent,
• Throughput – maximum number of tests nondisposable cuvets made of quartz glass. Other
generated per hour cuvets are made of plastic and are disposable
• Turnaround – amount of time to generate one result - Reaction temperatures and times vary for each
• Bar coding – mechanism for patient/sample analyte. The most common reaction temperatures are
identifications; used for reagent identification 37°C and 30°C
by an instrument - Kinetic assays – determination of sample
• Dead volume – amount of serum that cannot concentration is based on change in absorbance over
 time instrument. The program must use appropriate
- Endpoint/colometric assays – incubated for a standards and controls, statistical analyses, and a
specific time, absorbance determined, absorbance proficiency testing system
related to calibrators for calculation of sample - This information must be documented
concentration
- A spectrometer is built within the system to read
absorbances for kinetic and colometric assays
➢ These systems may use a diffraction
grating or aa series of high quality fibers.
Some automated analyzers incorporate
fluorometry or nephelometry
• Data Management
- The computer module of most automated
instruments has a data management system that
allows analysis of quality control (QC) materials
and assessment of patient values (e.g. delta check)
before releasing patient results
- Instruments/laboratory information systems
(LISs) also archive patient results and QC values.
These achieved results are stored by the
laboratory for various lengths of time

Point-of-Care Testing (POCT)

• Definition – performing diagnostic tests outside
the main laboratory and at or near patient care areas
• Applications – POCT is designed to provide
immediate laboratory test results for immediate
patient assessment and determination of
appropriate treatment
- POCT may be used in neonatal intensive care,
coronary care, intensive care or the emergency
department
• Operators
- Only waived laboratory tests can be performed
using POC instruments
- Clinical laboratory technicians and clinical
laboratory scientists must operate instruments
that perform complex or high-complexity
laboratory tests
• POC instrument evaluation
- All POC instruments must be evaluated in
accordance with the CLIA ‘88
- The values obtained from POC instruments
must correlate with values obtained from
larger laboratory instruments
- Linearity testing, calculation of control
ranges, correlations of sample data and
reference ranges must be done for each
instrument
• Training – all POC instrument operators must
be trained, and training must be documented
• Quality control – all effective quality control
systems must be set up for each POC
 • Qualified personnel
- Only properly certified personnel can perform
nonwaived assays
- They must be able to perform QC activities,
maintain instruments, and keep accurate and
systematic records of reagents and control
specimens, equipment maintenance, and
patient and analytical data
- They must have in-training service classes and
opportunities to attend a continuing education
program
Quality Assurance and Quality Nonanalytical Factors
- Periodic opportunities for personal upgrading
Control of technical skills and for obtaining new
Quality Assurance relevant information should be made available
to all persons working in the lab.
- A way of ensuring that - Personnel performance should be monitored
with periodic evaluations and reports.
quality requirements will • Established laboratory policies
- an updated manual contains a comprehensive
be fulfilled – the product listing of approved policies, acceptable
will be “fit for purpose” practices and precautions, including standard
blood and body fluid precautions
and made “right the first - should be included in a lab reference manual that is
available to all hospital personnel.
time”. - Each lab must have an up-to-date safety manual.
Contains comprehensive listing of approved
Quality Control
policies, acceptable practices, and standard
precautions (blood and body fluids).
- A process of fulfilling - OSHA and CDC
• Laboratory procedure manual
quality requirements by - this manual must be reviewed regularly by the
thoroughly reviewing the supervisor and updated, as needed.
- Should follow a specific pattern in how the
quality of all factors procedures are organized (CLSI).
- Minimal components:
involved in production. Title of the assay
Principle of the procedure & statement of clinical
Quality Assessment
application
• Currently, the majority of laboratory errors are
Protocol for specimen collection and storage
related to pre-analytical or post-analytical
Nonanalytical Factors
phases of testing. Specimen-related errors • QC information
continue to be a major problem, leading to • Reagents, supplies and equipment
unnecessary costs to hospitals • Procedural protocol
• To reduce and potentially eliminate laboratory • “Normal” reference ranges
errors, a quality assessment program is • Technical sources of error
• Limitations of the procedure
mandated
• Proper procedures for specimen collection and storage
• Two major components of quality assessment
program
- Non-analytical factors
- Analysis of quantitative data (quality control)

Non-analytical Factors
 regulations.
- Test request procedures, patient identification,
specimen procurement and labeling; specimen
transportation and processing procedures; lab
personnel performance; lab instrumentation, reagents
• Test questioning
and analytical test procedures; turn around times, and
- Lab test can be requested by a primary care
the accuracy of the final results.
provider or patient.
• Proficiency Testing (PT)
- the request, either hard copy or electronic,
- Identical samples are sent to a group of laboratories
must include all information about the test
participating in the PT program; each lab analyzes the
request, patient’s data accompanied by the
specimen, reports the results to the agency, and is
specimen to be tested
evaluated and graded on those results in comparison
- The information on the accompanying
to the results from other laboratories.
specimen container must match exactly the
• Interpretation of the Results of the Proficiency Testing
patient identification on the test request – (PT):
included in a database or handbook. - Each analyte has a define performance criteria
• Patient Identification, Specimen Procurement and
(example, +/-3SD of peer mean), where laboratories
Labeling using the same method are evaluated by comparing
- current information about obtaining appropriate
them with the group.
specimens, special collection requirements of - In external QC , difference of greater than 2SD in the
various types of tests, ordering tests correctly results indicates that a laboratory is not in agreement
and transporting and processing of specimens with the rest of the laboratories included in the
appropriately should be included in the database. program.
• Specimen Transportation and Processing
- If in case a clinical laboratory failed to identify or
- Some assays require special handling conditions,
resolve an error or discrepancy in the test process, the
such as placing the specimen on ice immediately facility is at risk of continuous operation and may be
after collection. recommended for closure.
• Accuracy in Reporting Results and Documentation:
- Specimens should be tested within 2 hours after - Many laboratories have established critical values or
collection to produce accurate results. the Delta check system to monitor individual patient
results.
- The documentation of specimen arrival times in - Highly abnormal individual test values and significant
the lab as well other specific test request data is differences from previous results in the Delta check
an important aspect of the quality assessment system alert the technologist to a potential problem.
process. - The ongoing process of making certain that the correct
• Preventive Maintenance of Equipment lab result is reported for the right patient in a timely
- Monitoring of the temperatures of the heat manner and at the correct cost is known as continuous
blocks and refrigerators is important in the quality improvement (CQI).
quality of test performance.
- Microscopes, centrifuges and other equipment
need regularly to be cleaned and checked for
accuracy.
- Mandatory recalibration of instrument systems.
• Appropriate Testing Methods
- Each lab must have an assessment routine for all
lab procedures, performed on a daily, weekly or
monthly basis to detect problems such as trends
and shifts in the established mean values.
- When such problems are indicated, they must be
corrected as soon as possible.
• Quality Assessment Procedures
- The documentation of an ongoing quality Quality Control (QC)
assessment program is mandated by CLIA
 • A system of ensuring accuracy & precision in the
lab by including quality control reagents in every
series of measurements.
• A process of ensuring that analytical results are
correct by testing known samples that resemble
patient samples.
• It involves the process of monitoring the
characteristics of the analytical processes and
detects analytical errors during testing and
ultimately prevent the reporting of inaccurate
patient test results.
• It is one component of the quality assurance
system and is part of the performance
monitoring test that occurs after a test has
been established.
• Consists of procedures used to detect error that
result from test system failure, adverse
environmental conditions, and variance in operator
performance, as
well as procedures to monitor the accuracy and
precision of test performance over time
• Accrediting agencies require monitoring and
documentation of quality assessment records
• QC activities include monitoring the performance
of laboratory instruments, reagents, other testing
products and equipment
• A written record of QC activities for each procedure
or function should include details of deviation from
the usual results, problems, or failures in functioning
or in the analytical procedure and any corrective
action taken in response to these problems
• Documentation of QC includes preventive
maintenance records, temperature charts, and QC
charts for specific assays
• All products and reagents used in the analytical
procedures must be carefully checked before
actual use in testing patient samples
• Use of QC specimens, proficiency testing and
standards depends on the specific requirements of
the accrediting agency
Parameters
• Sensitivity – ability of an analytical method to
measure the smallest concentration of the analyte of
interest.
• Specificity – ability of an analytical method to
measure only the analyte of interest
 - varies from sample to sample.
- basis for varying differences between repeated
measurements – variations in technique.
- due to instrument, operator, and environmental
conditions (variations in technique) such as pipetting
error, mislabeling of samples, temperature
fluctuation, and improper mixing of sample and
reagent.

2.Systematic Error
- error that influences observations consistently in one
direction (constant difference).
- detected as either positive or negative bias – often related
to calibration problems, deterioration of reagents and
control materials, improperly made standard solutions,
contaminated solutions, unstable and inadequate reagent
• Practicability - is the degree by which a method
blanks, leaky ion selective electrodes, failing instrumentation
is easily repeated.
and poorly written procedures.
• Reliability – the ability of an analytical method
to maintain accuracy and precision over an
extended period of time during which a. Constant Error
equipment , reagents, and personnel may - refers to the difference between the target value and the
change. assayed value.
Kinds of Quality Control - independent of sample concentration.
Intralab Quality Control (internal QC) - exists when there is continual difference between the
• involves the analysis of control samples together comparative method and the test method regardless of the
with the patient specimens. concentration.
• detects changes in performance between the
present operation and the “stable” operation.
• important for the daily monitoring of accuracy
and precision of analytical methods.
• detects both random and systematic errors in a
daily basis.
Interlab Quality Control (external QC)
• involves proficiency testing programs that
periodically provide samples of unknown
concentrations to participating clinical
laboratories.
• important in maintaining long-term accuracy of
the analytical methods.
• used to determine state-of-the-art
interlaboratory performance.
• the College of American Pathologist (CAP)
proficiency program is the gold standard for b. Proportional/Slope/Percent Error
clinical lab external QC testing. - results in greater deviation from the target value due to
Variations higher sample concentration.
Errors encountered in the collection, preparation and - exists when the difference between the test method and
measurement of samples, including transcription and the comparative method values is proportional to the
analyte concentration.
releasing of laboratory results.

Types of Variations 3. Clerical Error

1.Random Error - the highest frequency of clerical errors occurs with thee
- present in all measurements, it is due to use of handwritten labels and request forms.
chance.
 - incorrect incubation of solution

- equipment/instrument malfunction

- improper calibration of equipment/calibration error

Post-analytical Errors – occur after a test result is

generated.

- unavailable or delayed lab results

- long turnaround time

Pre-analytical Errors – occur prior to the testing - wrong transcription of the patient’s data and lab
process results

- incorrect patient identification - missing lab results

- improper patient preparation - lab results submitted to the wrong

physician/doctors who did not request
- incorrect specimen collection for the lab test

- mislabeled specimen

Predictive Values
- incorrect order of draw
To assess the predictive value (PV) for a test, the sensitivity,
specificity, and prevalence of the disease in the population
- incorrect use of tubes for blood collection being studied must be known.

- incorrect anticoagulant to blood ratio The prevalence of the disease is the proportion
of population who has the disease. The incidence of a
- improper mixing of blood and anticoagulant disease is the number of subjects found to have the disease
within a defined period such a year , in a population of
100,000.
- incorrect specimen preservation

- mishandled specimen (transport and storage)

A positive predictive value (PV) for a test indicates
the number of patients with an abnormal test result
- incorrectly interpreted/ordered lab test who have the disease, compared with all patients
with an abnormal result, as follows
- incomplete centrifugation Number of patients with disease
Positive PV = & with abnormal test results
Total number of patients with
- incorrect data log-in
abnormal test results

Positive PV
Analytical Errors – occur within the laboratory during = ________Truepositives______________
the testing process. True positives + False positives

- incorrect sample and reagent volume

A negative predictive value (PV) for a test indicates the CV = SD x 100
number of patients with a normal test result who do
not have the disease, compared with all patients with a Mean
normal (negative) result, as follows

Negative PV = _ ___True negatives_________

True negatives + False negatives *Variance is called the standard deviation squared;
a measure of variability. It represents the difference
between each value and the average of the data.

V = (SD)2

Quality Control Chart

It is used to observe values of control materials over

time to determine reliability of the analytical
method.

It is utilized to observe and detect analytic errors

such as inaccuracy and imprecision.

1. Gaussian Curve (Bell-Shaped Curve)

*it occurs when the data set can be accurately

described by the SD and the mean.

*it is obtained by plotting the values from multiple

Statistics analyses of a sample.
It is the science of gathering, analyzing,
interpreting and presenting data. *it is a population probability distribution that is
symmetric about the mean.
*mean – is a measure of central tendency . It is
associated with symmetrical or normal *it occurs when data elements are centered around
distribution. the mean with most elements close to the mean.
mean = Σx n

*Standard Deviation (SD) – is a measure of the *it focuses on the distribution of errors from the
dispersion of values from the mean. It helps analytical method rather than the values from a
describe the normal curve. A measure of the healthy or patient population.
distribution range the most frequently used
measure of variation. *the total area under the curve is 1.0 or 100%

* Coefficient of Variation (CV) is a percentile 2. Cumulative Sum Graph (CUSUM)

expression of the mean; an index of precision.
*it calculate the difference between QC results *it easily identifies random and systematic errors.
and the target means.

*Common method: V mask

*it identifies consistent bias problems; it requires

computer implementation.

*this plot will give the earliest indication of

systematic errors (trend)and can be used with
the 135 rule.

*it is very sensitive to small, persistent errors that Errors which can be observed in L-J chart
commonly occur in the modern, low a. Trend
calibration-frequency analyzer.
it is formed by control values that either increase or
*results are out of control when the slope decrease for six consecutive days.
exceeds 45o or a decision ± 2.7 SD IS
exceeded. Main cause Deterioration of reagents

3. Youden/Twin Plot b. Shift

it is formed by control values that distribute
themselves on one side or either side of the mean for
*it is used to compare results obtained on a
six consecutive days.
high and low control serum from different
laboratories.
Shift in the reference range is due to transient
instrument differences.
*it displays the results of the analyses by plotting
the mean values for one specimen on the Main cause: Improper calibration of the instrument.
ordinate y-axis and the other specimen on the
abscissa x-axis.
c. Outliers

*the points falling from a center but on the 45o

*These are control values that are far from the main
line suggest a proportional error and points
set of values.
falling from the center but not on the 45o line
suggest a constant error.
*These are highly deviating values.
4. Shewhart Levey-Jennings Chart
*These are caused by random or systematic errors.
*it is the most widely used QC chart in the
clinical laboratory.
5. Westgard Control Chart
*it allows the laboratorians to apply multiple
rules without the aid of the computer. It recognizes that the use of simple upper and lower
control limits is not enough to identify analytical
*it is a graphic representation of the problems.
acceptable limits of variation in the results of an
analytical method. In Westgard, error detection rates can increase
without increasing the false rejection rate.
Westgard used the term control rule to indicate Lean System
if the analytical process is out of control. It is a system for reducing waste (“nonvalued
activities”)especially in production or manufacturing
Quality Control Chart processes.
*Six Westgard rules are
It was conceptualized to improve the automobile
typically used: industry in terms of the quality and efficiency of
automobile production.
three are warning rules and
It focuses on work flow actions in performing specific
three are mandatory rules. tasks, procedures, or other activities accomplished
by critically reviewing each step in the process to
determine where inefficiencies can be eliminated.

A “Lean Clinical Laboratory” utilizes fewer resources,

reduces costs, enhances productivity, promotes
staff morale, and improves the quality of patient
care

Ex. Relocating analytic equipment to an area that

would require fewer steps, thus improving
turnaround time, consolidating test panels to fewer
instruments, eliminating the expense of maintaining
multiple instruments and supplies, accesability to
instruments and materials such as pipettes, culture
tablets, reagents, etc. and relocating staff to
maximize and minimize wasteful down time.
Westgard Rules

accept the results of the testing run, if only a

warning is violated.

reject the results of the testing run, when a

mandatory rule is violated.

increase the retesting range for a particular

assay, if either a warning or mandatory rule is
violated.

Six Sigma (6σ)

It measures the degree of variability or error in

products or services through statistics and
quantitative parameters – process defects or
errors analyzed, potential causes are identified
and improvements are implemented.

Main Goal: To reduce the number of defects to

near zero.
Carbohydrates • Eat too many grams of carbohydrates for the amount of
insulin you took, or eat too much carbs in general
• Have an infection
Blood Sugar • Are ill
• Blood sugar or blood glucose • Are under stress
- Refers to sugar that is transported • Become inactive or exercise less than usual
through the bloodstream in order to • Take part in strenuous physical activity, especially when
supply energy to all the cells in our bodies you blood sugar levels are high and insulin levels are low
- The sugar is made from the food we eat
- The human body regulates blood glucose Symptoms (Early Signs)
levels so that they are neither too high nor • Increased thirst
too low • Headache
• Sugar • Trouble concentrating
- Is a simple, crystalline, edible • Blurred vision
carbohydrate and comes in a variety of • Frequent peeing
forms, all of the sweet • Fatigue (weak, tired feeling)
- Our body digests carbohydrates into • Weight loss
• Blood sugar more than 180mg/dl
glucose, a simple sugar than can easily
convert to energy
- The chemical formula for glucose is C5H12O6
How does the body control glucose in the blood?
• The human digestive system breaks down the • The normal blood glucose level ranges between 3.5-
carbohydrates from food into various sugar 7.8mmol/L.
molecules • Insulin – lowers; Glucagon – heightens
– one of them is glucose, the body’s principal
source of energy. The glucose goes straight
from the digestive system into the
bloodstream after we have consumed and
digested food. Glucose can only enter cells if
there is insulin in the bloodstream too.
Without any insulin the cells would starve
• After we eat, blood sugar concentrations rise,
the pancreas releases insulin automatically so
that the glucose enters cells, as more and
more cells receive glucose, blood sugar levels
come down to normal again. Excess glucose is
stored as glycogen (stored glucose) in the liver The Glucose Journey (From food to fuel via bloodstream)
and muscles 1. Glucose starts its journey as a carbohydrate food
• After a meal blood glucose levels rise, insulin 2. When the food is eaten, it passes through the mouth,
is released from the pancreas into the stomach and small intestine
bloodstream, blood sugar levels fallback - All of these areas help to digest (break down) the
• If you have not eaten for a while and blood food to glucose
glucose concentrations keep dropping, the 3. The glucose is absorbed from the small intestine into the
pancreas releases another hormone called bloodstream
glucagon 4. The bloodstream carries the glucose to its next stop, all
• Glucagon the body’s cells, particularly muscles, the brain and the
- Triggers the breakdown of glycogen into liver
glucose, thus pushing blood glucose - Your brain won’t function well if your glucose is low
levels back up to normal 5. The glucose can only enter the cells with the help of
insulin, a hormone which is made in the pancreas
Causes of High Blood Sugar
Your blood sugar may rise 6. As the blood glucose level rises after eating, the
• Skip or forget your insulin or oral glucose pancreas releases insulin into the bloodstream. Insulin
lowering medicine travels to the cells, where it works to allow glucose to
enter the cells
7. Glucose is also directed to the liver to be stored for - Increases glucose absorption from the intestines
later use. Between meals and overnight our body
can draw on the stored glucose for energy.
• During a fast, the blood glucose level is kept Renal Threshold for Glucose
constant by mobilizing the glycogen stores in the • Glucose is filtered by the glomeruli, reabsorbed by
liver the tubules, and normally not present in the urine
• During long fasts, gluconeogenesis is required to • If the blood glucose level is elevated, glucose
maintain blood glucose levels because glycogen appears in the urine, a condition known as
stores are used up in about 24 to 48 hrs glucosuria
• An individual with a fasting blood glucose level • An individual’s renal threshold for glucose varies
- > 100mg/dL is referred to as hyperglycemic between 160 and 180 mg/dl
• An individual with a fasting blood glucose level - When blood glucose reaches this level or exceeds
- < 50mg/dL is referred to as hypoglycemic. it, the renal tubular transport mechanism
becomes saturated, which causes glucose to
Hormones Affecting Blood Glucose be excreted into the urine.
• Insulin • In severe hyperglycemia, one has polyuria,
ketonuria and glycosuria
- Produced by the beta cells of the
- Polyuria – frequenturinating
pancreatic islets of Langerhans
- Ketonuria – ketone bodies in the urine
- Promotes the entry of glucose into the
- Glycosuria – glycogen in the urine
liver, muscle, and adipose tissue to be
• Ingestion of ethanol, salicylate and propanol may
stored as glycogen and fat
cause hypoglycemia
- Inhibits the release of glucose form the liver
• Somatostasin
- Synthesized by delta cells of the
Symptoms of Diabetes
pancreatic islets of Langerhans
• Always tired
- Inhibits secretion of insulin, glucagon, • Frequent urination
and growth hormone, resulting in an • Sudden weight loss
increase in plasma glucose level • Wounds that won’t heal
• Growth Hormone and the • Sexual problems
Adrenocorticotropic Hormone (Acth) • Always hungry
- Hormones secreted by an anterior pituitary • Blurry vision
that arise blood glucose levels • Numb or tingling hands or feet
• Always thirsty
• Cortisol • Vaginal infections
- Secreted by the adrenal glands
- Stimulates glycogenolysis, lipolysis and
Diabetes
gluconeogenesis
• Describes a group of metabolic diseases in which the
• Epinephrine
person has high blood glucose (blood sugar), either
- Secreted by the medulla of the adrenal glands
because insulin production is inadequate, or because the
- It stimulates glycogenolysis and lipolysis
body’s cells do not respond properly to insulin, or both
- It inhibits secretion of insulin
• Pancreas produce insulin according to the blood
- Physical or emotional stress causes
glucose level
increased secretion of epinephrine and an
- Without the pancreas, a person can die
immediate increase in blood glucose levels
• Pathophysiology
• Glucagon
- General
- Secreted by the alpha cells of the
➢ Insulin – is the principal hormone that regulates
pancreatic islets of Langerhans
the uptake of glucose from the blood into cells of
- Increases blood glucose by stimulating
the body, especially liver, adipose tissue and
glycogenolysis and gluconeogenesis
muscle, except smooth muscle, in which insulin
• Thyroxine
- Secreted by the thyroid gland
acts via the IGF-1 (insulin-like growth factor-1)
➢ Therefore, deficiency of insulin or the
- Stimulates the glycogenolysis and
gluconeogenesis insensitivity of its receptors plays a central role
in all forms of diabetes mellitus
 ➢ The body obtains glucose from 3 main places: cell)
o The intestinal absorption of food ➢ This type can be further classified as
o The breakdown of glycogen, the immune-mediated or idiopathic
storage form of glucose found in the ➢ The majority of Type 1 Diabetes is of the
liver immune-mediated nature, in which a T-cell
o Gluconeogenesis, the generation of mediated autoimmune attach leads to the loss of
glucose from non-carbohydrate beta cells and this insulin
substrates in the body
o T-cell is the one attacking the beta cells that
➢ Insulin plays a critical role in balancing produces insulin
glucose levels in the body: ➢ Most affected people are otherwise healthy
o It can inhibit the breakdown of and of a healthy weight when onset occurs
glycogen or the process of ➢ Sensitivity and responsiveness to insulin are
gluconeogenesis
usually normal, especially in the early stages
o It can stimulate the transport of
➢ Type 1 Diabetes can affect children or adults,
glucose into fat and muscle cells
but was traditionally termed “juvenile
o It can stimulate the storage of glucose
in the form of glycogen diabetes” because a majority of these diabetes
➢ Insulin is released into the blood by beta cells, cases were in children
➢ Type 1 Diabetes is partly inherited, with
found in the islets of Langerhans in the
pancreas, in response to rising levels of multiple genes, including certain HLA
blood glucose, typically after eating genotypes, known to influence the risk of
o Lower glucose levels result in
diabetes.
o Possibility to have diabetes so it is
decreased insulin release from the beta
cells and results in the breakdown of somewhat a marker or receptor that you
glycogen to glucose are at risk of diabetes
➢ In genetically susceptible people, the onset of
o This process is mainly controlled by the
hormone glucagon, which acts in the diabetes can be triggered by one or more
opposite manner to insulin environmental factors, such as a viral infection
➢ If the amount of insulin available is or diet
insufficient, if cells respond poorly to the ➢ Among dietary factors, gluten may lead to type
effects of insulin, if the insulin itself is 1 diabetes, but the mechanism is not fully
defective, then glucose will not be understood
absorbed properly by the body cells, the
net effect is persistently high levels of - Type 2 Diabetes Mellitus
blood glucose, poor protein synthesis, and ➢ Type 2 DM is characterized by insulin
breakdown of fat storage – Acidosis resistance
➢ When the glucose concentration in the ➢ The defective responsiveness of body tissues to
blood remains high over time, the kidneys insulin is believed to involve the insulin
will reach a threshold of reabsorption -> receptor
Glycosuria -> (this increases the osmotic ➢ In the early stage of type 2, the predominant
pressure of the urine) -> polyuria -> abnormality is reduced insulin sensitivity
(increased fluid loss) -> lost blood volume ➢ Type 2 DM is due to primarily to lifestyle
will be replaced osmotically from water factors and genetics
held in body cells and other body - A number of lifestyle factors are known to be important
compartments -> dehydration -> to the development of type 2 diabetes, including:
polydipsia ➢ Obesity
➢ Lack of physical activity
- Type 1 Diabetes Mellitus ➢ Poor diet
➢ Stress
➢ Type 1 Diabetes Mellitus is characterized
- Dietary factors also influence the risk of developing
by loss of the insulin-producing beta cells
type 2 diabetes such as:
of the islets of Langerhans in the
➢ Sugar-sweetened drinks
pancreas, leading to insulin deficiency ➢ Type of fats in diet
➢ Glucose will go to the cell to release insulin o Saturated fats and trans fatty acids
(it serves as the key for glucose to enter the
 increasing the risk
o Polyunsaturated
and monounsaturated fat • Oral Manifestations andComplications
decreasing the risk - Periodontal changes are seen in Diabetes Mellitus
➢ Eating lots of white rice also may increase the - Salivary glands
risk of diabetes ➢ Xerostomia is common, but reason is unclear
➢ A lack of exercise is believed to cause 7% of ➢ Tenderness, pain and burning
cases sensation of tongue
- Gestational Diabetes - Dental caries
➢ Gestational Diabetes Mellitus (GDM) ➢ Increase caries prevalence in adult
resembles type 2 diabetes in several aspects with diabetes (xerostomia, increase
➢ Involves a combination of relatively saliva glucose)
inadequate insulin secretion and
responsiveness Feature Type 1 Type 2
➢ It occurs in about 2-10% of all pregnancies Diabetes Diabetes
Onset Sudden Gradual
and may improve or disappear after delivery
Age at onset Mostly in Mostly in adults
➢ However, after pregnancy approx. 5-10% of
children
women with gestational diabetes are found Body Size Thin or normal Often obese
to have diabetes mellitus, most commonly Ketoacidosis Common Rare
type2 Autoantibodies Usually present Absent
➢ Gestational diabetes is fully treatable, but Endogenous Low or absent Normal,
requires careful medical supervision Insulin decreased or
throughout the pregnancy increased
➢ Management may include dietary changes, Concordance in 50% 90%
identical twins
blood glucose monitoring, and in some Prevalence ~ 10% ~ 90%
cases, insulin may be required
➢ Hyperglycemia state shows a
➢ Through it may be transient, untreated
positive association with dental caries
gestational diabetes can damage the health
➢ Carious Lesion on teeth with Xerostomia.
of the fetus or mother
May cause secondary enlargement of
➢ Risks to the baby include:
parotid glands with Sialosis
o Macrosomia (high birth weight) – high
blood glucose levels in the mother
• Comparison of Type 1 and 2 Diabetes
Brings extra glucose to baby Causes
baby to put on extra weight
o Congenital heart defects How are Diabetes and Pre-Diabetes Diagnosed?
o Central nervous system abnormalities • Blood tests are used to diagnosis diabetes and
o Skeletal musclemalformations
pre- diabetes. Lab analysis of blood is needed to
➢ Increased levels of insulin in a fetus’ blood
ensure test results are accurate
may inhibit fetal surfactant production and
• Glucose measuring devices used in a health
cause respiratory distress syndrome
➢ A high blood bilirubin level may result from
care provider’s office, such as finger-stick
devices, are not accurate enough for diagnosis
RBC destruction (hemolysis) but may be used as a quick indicator of high
➢ In severe cases, perinatal death may occur, blood glucose
most commonly as a result of poor • Patients with Type 1 Diabetes will need to take
placental perfusion due to vascular insulin injections for the rest of their life. They
impairment must also ensure proper blood-glucose levels
➢ Labor induction may be indicated with by carrying out regular blood tests and
decreased placental function. following a special diet
• Type 1 Diabetes is usually diagnosed in children
➢ A Caesarean section may be performed
and young adults. Only 10% of people with
if there is marked fetal distress or an
diabetes have this form of the disease
increased risk of injury associated with • In type 1 diabetes, the body does not produce insulin
macrosomia, such as shoulder dystocia
 Types of Diabetes stream, and differing color responses of the
indicator strip reflect glucose concentration
• While type 1 and type 2 are the most ➢ Benedict’s and Fehling’s test can also detect
common form of diabetes, there are glucosuria
others that you may hear about. Impaired • Ketonuria
Glucose Metabolism or Pre-diabetes - Qualitative detection of ketone bodies can be
• There are two pre-diabetes conditions: accomplished by nitroprusside tests (Acetest or
- Impaired Glucose Tolerance (IGT) Ketostix), Rothera’s test, etc
is where blood glucose levels are - These tests do not detect Beta-hydroxyl butyric
higher than normal but not high acid, which lacks a ketone group
enough to be classified as diabetes - Ketone bodies may be resent in a normal subject as a
- Impaired Fasting Glucose (IFG) is where result of simple prolonged fasting
blood glucose levels are escalated in the • Microalbuminuria
fasting state but not high enough to be - May be defined as an albumin excretion rate
classified as diabetes intermediate between normality (2.5-25mg/day) and
• On the other hand, despite eating so often and a lot, macroalbuminuria (250mg/day)
the patients lose weight - The small increase in urinary albumin excretion is not
• With the usage of protein as energy source, the detected by simple albumin stick tests and requires
patient feels themselves tired and sluggish confirmation by careful quantization in a 24hour
- If blood glucose is too high, it is tried to be urine specimen
thrown away by kidneys so the patients begin - The importance of microalbuminuria in the
to urinate so often diabetic patient is that it is a signal of early
- As a result, the patients feel thirsty and start to reversible renal damage
drink a lot - Performing an albumin-to-creatinine ratio is
probably easiest
What happens if there is a problem with the production of - Microalbuminuria is a common finding (even at
insulin? diagnosis) in type 2 diabetes mellitus and is a risk
• Glucose in blood is not able to go into the cell factor for macro vascular (especially coronary
• The cells can’t meet energy needs and energy is heart) disease.
tried to be provided from fat and protein
• Using fat as the energy source results the increasing
of ketone in the body Blood Chemistry
• Blood Glucose Estimation
- Choice of Sample
Urine Analysis ➢ Plasma or serum from venous blood
• Detection of Urinary Glucose (Glucosuria) samples has the advantage over whole
- First-line screening test for diabetes mellitus blood of providing values for glucose that
- Normally, glucose does not appear in urine until are independent of Hematocrit and that
the plasma glucose rises above 160–180mg/dL reflect the glucose concentration to which
- In certain individuals due to low renal threshold body tissues are exposed
glucose may be present despite normal blood ➢ Plasma and serum are more readily
glucose levels measured on automated equipment, they
- Conversely renal threshold increases with age are used in most laboratory
so many diabetics may not have glycosuria ➢ If serum is used or plasma is collected from
despite high blood sugar levels tubes that lack an agent to block glucose
- Detection of glucosuria metabolism (such as fluoride), samples
➢ A specific and convenient method to should be refrigerated and separated
detect glucosuria is the paper strip within 1 hour after collection
impregnated with glucose oxidase and a ➢ The glucose concentration is 10-15% higher
chromogen system (Clinistix, Diastix), in plasma or serum than in whole blood
which is sensitive to as little as 0.1% glucose because structural components of blood
in urine are absent
➢ Diastix can be directly applied to the urinary - Fasting Blood Glucose
 ➢ Is measured after an overnight fast of - Since glycohemoglobin circulate within red blood
10 hours cells whose reflect the state of glycemia over the
➢ FBG estimation is better than random preceding 8-12 weeks, thereby providing an
blood glucose improved method of assessing diabetic control
o Normal: FPG < 5.6 mmol/L (100mg/dL) ➢ We will know the stored sugar in the body
o IFG: FPG = 5.6 to 6.9 mmol/L (100 to within 3 months so they make use this fraction
125 mg/dL) of hemoglobin to monitor diabetes
o Warrants the diagnosis of DM: ➢ Monitored for 3 months
FPG >7.0 mmol/L (126mg/dL) - Any condition that shortens erythrocyte survival or
- Random Blood Glucose decreases mean erythrocyte age (e.g., Recovery from
➢ Random is defined as without regard acute blood loss, hemolytic anemia) will falsely
to time since the last meal lower HbA1C irrespective of the assay method used
➢ RBG measurement is required only - Methods for measuring HbA1C include:
during emergency ➢ Electrophoresis
➢ The current criteria for the diagnosis ➢ Cation-exchange chromatography
of DM emphasize that the FPG is the ➢ Boronate affinity chromatography
most reliable and convenient test for ➢ Immunoassays
identifying DM in symptomatic
individuals • Serum Fructosamine Estimation
➢ A random plasma glucose concentration - Serum fructosamine is formed bynonenzymatic
>11.1 mmol/L (200 mg/dL) glycosylation of serum proteins (predominantly
accompanied by classic symptoms of albumin)
DM (polyuria, polydipsia, weight loss) ➢ Pregnant women make use of fructosamine
is sufficient for the diagnosis of DM estimation
- Glucose Tolerance Test - Serum albumin has a much shorter half-life than
➢ When the fasting plasma glucose level hemoglobin, serum fructosamine generally reflects
is 125mg/dl or higher on more than the state of glycemic control for only the preceding 1-
one occasion, further evaluation of 2 weeks
the patient with a glucose challenge is - Normal values vary in relation to the serum, albumin
unnecessary concentration are 1.5-2.4 mmol/L when the serum
➢ However, when fasting plasma glucose is albumin level is 5g/dL
less than 126 mg/dl in suspected cases, a - When abnormal hemoglobin or hemolytic states
standardized oral glucose tolerance test affect the interpretation of glycohemoglobin or
may be done when a narrower time frame is required, such as for
➢ Methodology: 75g of glucose dissolved in ascertaining glycemic control at the time of
300mL of water is given after an overnight conception in a diabetic woman who has recently
fast to a person who has been receiving at become pregnant, serum fructosamine assays offer
least 15-0-200g of carbohydrate daily for 3 some advantage
days before the test • Self-Monitoring of Blood Glucose
➢ Data Interpretation: The Diabetes Expert - Capillary blood glucose measurements
Committee criteria for evaluating the performed by patients themselves, as
standard oral glucose tolerance test outpatients, are extremely useful
Normal Impaired - In type 1 patients in whom “tight” metabolic
Diabetes control is attempted, they are indispensable
glucose glucose
Mellitus - There are several paper strips (glucose
tolerance tolerance
Fasting plasma oxidase, glucose dehydrogenase, or
glucose < 110 110 – 125 > 126 hexokinase) methods for measuring glucose on
(mg/dL) capillary blood samples
2 hrs after
< 140 140 – 199 > 200
glucose load
• Lipid Profile
- Serum total cholesterol is elevated
• Glycated Hemoglobin (HbA1C)Measurements - Serum triglycerides are high
- HbA1C comprises 4-6% of total hemoglobin A1 - Serum HDLc is low
 - Qualitative change in LDL particles, glucose utilization and storage
producing a smaller dense particle whose - Insulin Preparations
membrane carries supranormal amounts ➢ Insulin is indicated for type 1 diabetes as well as
of free cholesterol for type 2 diabetic patients with insulinopenia
- These smaller dense LDL particles are whose hyperglycemia does not respond to diet
more susceptible to oxidation, which therapy either alone or combines with other
renders them more atherogenic hypoglycemic drugs
➢ With the development of highly purified human
• Additional Tests insulin preparations, immunogenicity has been
- The patient should be screened for DM-
markedly reduced, thereby decreasing the
associated conditions (e.g., Kidney, liver incidence of insulin allergy, immune insulin
and thyroid dysfunction) resistance and localized lipoatrophy at the
- Individuals at high risk for cardiovascular
injection site
disease should be screened for - Methods of InsulinAdministration
asymptomatic CAD by appropriate cardiac ➢ Insulin syringes and needle
stress testing, when indicated o Plastic disposable syringes are available in
- The classification of the type of DM may 1mL, 0.5mL and 0.3mL sizes
be facilitated by laboratory assessments ➢ Insulin pen injector devices
• Serum insulin or C-peptide measurements do not o Insulin pens eliminate the need for carrying
always distinguish type 1 from type 1 DM, but a low C- insulin vials and syringes
peptide level confirms a patient’s need for insulin ➢ Insulin pumps
o Insulin infusion pumps are used for
Immunological Assays subcutaneously delivery of insulin. These
• Antibodies to insulin, islet cells, or Glutamic
pumps are small (about the size of a pager)
acid decarboxylase (GAD) can be estimated to and very easy to program
o It is an alternative to multiple daily injections
differentiate between the types of diabetes
of insulin by insulin syringe or an insulin pen
mellitus
• They are absent in type 2 diabetes mellitus
and allows for intensive insulin therapy
• Latent autoimmune diabetes of adults, or when used in conjunction with blood glucose
LADA, is a form of slow-onset type 1 diabetes monitoring.
➢ Inhaled insulin
that occurs in middle-aged (usually white)
o A novel method for delivering a pre- prandial
adults
• It can be differentiated from type 2 diabetes
powdered form of insulin by inhalation
by measuring anti-GAD65 antibodies (Exubera) has been approved by the FDA
o The FDA approved the first inhaled version
of insulin called Exubera from Pfizer Inc.
Management of Diabetes Mellitus
- Complications of InsulinTherapy
• Eliminate symptoms related to hyperglycemia
➢ Hypoglycemia, insulin allergy, immune insulin
• Reduce or eliminate the long-term
resistance and lipodystrophy at the injection site
microvascular and macrovascular
are some of the complications of insulin therapy
complications of DM
➢ Besides insulin therapy, lifelong dietary and
• Allow the patient to achieve as normal a
lifestyle modifications are required to be done to
lifestyle as possible
• Management of Type 1 Diabetes Mellitus
achieve euglycemia
- Islet Cell Transplantation
- Because individuals with type 1 DM
➢ Is minimally invasive procedure and wide
partially or completely lack endogenous
application of this procedure for the treatment
insulin production, administration of
of type 1 diabetes is limited by the dependence on
basal, exogenous insulin is essential for
multiple donors and the requirement for potent
regulating glycogen breakdown,
long-term immunotherapy
gluconeogenesis, lipolysis and
- Stem Cell Therapy
ketogenesis ➢ Is one of the most promising treatments for the
- Likewise, insulin replacement for meals
near future. It is expected that this kind of therapy
should be appropriate for the can ameliorate or even reverse some diseases
carbohydrate intake and promote normal
 autoimmune destruction of insulin producing beta
cells of the pancreas
• MANAGEMENT OF TYPE 2 DIABETES MELLITUS • The classical symptoms of type 1 DM are polyuria,
- The goals of therapy for type 2 DM are similar
polydipsia, polyphagia and weight loss
to those in type 1 • Is fatal unless treated with insulin
- The care of individuals with type 2 DM • Injection is the most common method of
must also include attention to the administering insulin; insulin pumps and inhaled
treatment of conditions associated with insulin has been available at various times
type 2 DM (obesity, hypertension, • Diabetic keto-acidosis is the most common
dyslipidemia, cardiovasculardisease) complication of type 1 DM
- Detection or management of DM-related
complications Summary of Type 2 DM
• Is characterized by impaired insulin secretion,
- Weight Reduction insulin resistance, excessive hepatic glucose
➢ Treatment is directed toward production, and abnormal fat metabolism
achieving weight reduction, and • While many patients with type 2 diabetes present
prescribing a diet is only one means to with increased urination and thirst, many other
this end have an insidious onset of hyperglycemia and are
➢ Behavior modification to achieve asymptomatic initially
adherence to the diet • Hyperglycemic hyperosmolar state (HHS) is an
➢ Increased physical activity to expend energy acute complication of type 2 DM. chronic
– complications are micro and macro vascular
is also required involving small and large blood vessels
- Hypoglycemic Agents respectively.
➢ If the patient is not able to achieve
• Glucose lowering agents that either increase insulin
target glycemic control with weight
secretion, reduce glucose production, increase insulin
management and exercise, then
sensitivity, and enhance GLP-1 (Glucagon like peptide)
pharmacologic therapy is indicated
action are used to treat hyperglycemia
➢ Based on their mechanisms of action,
• The care of individuals with type 2 DM must also include
glucose- lowering agents are
attention to the treatment of conditions associated
subdivided into agents that increase
with type 2 DM (obesity, hypertension, dyslipidemia,
insulin secretion, reduce glucose
cardiovascular disease) and detection or management of
production and increase insulin
DM-related complications
sensitivity

• Biguanides
- Metformin is representative of this class of
agents
- It reduces hepatic glucose production
through an undefined mechanism and
improves peripheral glucose utilization
slightly
- Metformin reduces fasting plasma
glucose and insulin levels, improves the
lipid profile and promotes modest weight
loss
- The major toxicity of metformin, lactic
acidosis, can be prevented by careful
patient selection

Summary of Type 1 DM
• Type 1, IDDM, or juvenile diabetes – is a form
of diabetes mellitus that results from
 Children with type 1 diabetes will need to take insulin
for the rest of their lives, unless a cure in found one
Estimation of blood glucose day
• Measurement of blood glucose is indicative of • Only older people develop type 2 diabetes – things are
current state of carbohydrate metabolism. changing.
• Depending on time of collection: - A growing number of children and teenagers are
developing type 2 diabetes due to the explosion in
o Fasting blood glucose-after an overnight
childhood obesity rates, poor diet, and physical
fast.
inactivity
o Post meal or postprandial blood • If you have diabetes, you cannot eat chocolates or
glucose-2 hrs after the subject has taken sweets – people with diabetes can eat chocolates and
a normal meal. sweets if they combine them with exercise or eat them as a
o Random blood glucose - Any time of the part of a healthy meal
day. • Diabetic patients cannot eat bread, potatoes or pasta –
people with diabetes can eat starchy foods. However,
Laboratory test for diagnosis
they must keep an eye on the size of the portions
1. Estimation of blood glucose. • Diabetes diets are different from other people’s – the
diet doctors recommend healthy nutrition; healthy for
2. Oral glucose tolerance test. everybody. Meals should contain plenty of vegetables,
fruit, whole grains, and they should be low in salt and
sugar, and saturated or trans-fat.

What can be done for diabetes at school?

• Brochures and films should be prepared to inform the
students about diabetes
• Students should be informed about the importance of
healthy eating and doing exercises
• School canteens should be controlled and warned
to sell healthy food and healthy drinks rather than
fast food and fizzy drinks
• Students should be informed about not eating fast
food
Diet Plan • Parents should be informed about healthy
• Daily nutritional needs should be taken frequently nutrition and the importance of homemade food in
but small portions children’s bag
• Teachers should follow their students about their
Diabetes Myths health problems. If they have some symptoms with
• People with diabetes should not exercise – NOT any disease, they should contact the parents
TRUE! • They should also inform the students about the
- Exercise is important for people with diabetes, importance of their health.
as it is for everybody else
- Diabetic patients should discuss exercise with
their doctors before starting the exercise
• Fat people always develop type 2 diabetes
eventually – this is not true.
- Being overweight or obese raises the risk of
becoming diabetic, they are risk factors, but do
not mean that an obese person will definitely
become diabetic
• Children can outgrow diabetes – this is not true.
- Nearly all children with diabetes have type 1;
insulin-producing beta cells in the pancreas
have been destroyed. These never come back.