CC Lec - Prelim Exam

CLINICAL CHEMISTRY LEC
LECTURE 1: LABORATORY SAFETY AND

PATIENT PREPARATION
Prof. JC Louise Bandala, RMT
August 17, 2021
For updates and corrections → @mar4rii on Twitter
Laboratory Safety - rules used in every lab to keep everyone safe ● Focuses on safety related to
blood-borne pathogens that
LABORATORY SAFETY AND REGULATIONS can be present
● Safety Agencies and Organizations
○ U.S. Department of Labor’s Occupational Safety Awareness for Clinical Lab. Personnel
Safety and Health Administration (OSHA)
○ Clinical and Laboratory Standards Institute Employer’s Responsibilities Employee’s Responsibilities
(CLSI)
○ CDC, part of the U.S. Department of Health and Establish lab work methods & Know and comply with the
Human Services (DHHS), Public Health Service safety policies established lab works safety
○ College of American Pathologists (CAP) methods
○ The Joint Commission (The Joint Commission Provide supervision & guidance Have a positive attitude toward
on Accreditation of Healthcare Organization) to employees supervisors, coworkers, facilities
■ Formerly known as JCAHO & safety training
● Each of these organizations has its own rules Provide safety info, training, Give prompt notification of
● CLSI - formerly known as NCCLS (National Committee PPE & medical surveillance to unsafe conditions or practices to
for Clinical Laboratory Standards) employees the immediate supervisor and
○ Provides excellent general laboratory safety ensure that unsafe conditions
and infection control guidelines and practices are corrected
○ They already created 2 documents
■ The first is about clinical laboratory Provide and maintain equipment Engage in the conduct of safe
safety and lab facilities that are work practices and use of PPE
■ Second is the protection of laboratory adequate for the tasks required
workers from occupationally acquired ● Employer and employee should share safety
infections responsibility
● CAP - publishes an extensive inspection checklist as ○ The individual employee has on obligation to
part of the lab accreditation program which includes follow safe work practices, and be attentive to
section dedicated to lab safety potential hazards in the place their working at
● TJC - publishes a yearly accreditation manual for ○ Employers has the ultimate responsibility for
hospitals and the accreditation manual pathology and safety and delegates authority for safe
clinical laboratory services which actually includes a operation to laboratory managers and
detailed section on safety requirements supervisors
● If we summarize their roles, it's basically more on setting ○ No matter how careful we are, if the workplace
guidelines and accreditation of different laboratories in itself do not have the correct policies while
relation to if it reached the required safety present in a working = it disregards the safety
workplace ● Being an employer, you are liable to your employees.
○ Whatever may happen to employees means
Occupational Safety and Health Act that the workplace is harmful
● Public Law 91-596 ● Employees should be aware and should understand how
● Enacted by US Congress in 1970 everything works
● Goals: Provide all employees with a safe work ○ Identify the different threats (electrocution, etc.)
environment
● OSHA (Occupational Safety and Health Administration) Laboratory Hazards Prevention Strategies
○ Inspection ● Engineering Controls
- Authorized to conduct on-site ● Administrative Controls
inspections to determine whether an ● Work Practice Controls
employer is complying with the ● Personal Protective Equipment (PPE)
mandatory standards in terms of
safety OSHA’s Three Lines of Defense
- Safety is no longer a moral obligation
but also a federal law
○ Accreditation
● There are a lot of standards under OSHA
○ There are different standards that is explained
■ Blood-borne pathogen standard
■ Hazard communication standard
■ Respiratory protection standard
○ Basically, it pertains to safety specific to those
standard
■ Ex. blood-borne pathogen standard
1
● Hierarchy of Controls Color of container/bag Type of waste
○ Elimination Black Non-infectious dry waste
■ Most effective way to ensure safety
Green Non-infectious wet waste (kitchen, dietary,
■ To completely remove the hazard and
etc.)
if not possible the next thing we can
do is substitution Yellow Infectious and Pathological waste
○ Substitution Yellow with black band Chemical waste including those with heavy
■ Replace the hazard or at least find metals
safer alternatives to those existing
hazards Orange Radioactive waste
○ Engineering controls Red Sharps and pressurized containers
○ Administrative controls
Purple Cytotoxic or cytostatic waste must be
○ PPE
incinerated in a licensed or permitted facility
4. Splash guards
5. Volatile liquid carriers
- Designed to keep intact chemicals
6. Centrifuge safety buckets
● Example on how we plan and ensure safety to the

workplace
● Ex. for people checking packages
○ Involves the use of utility knives
■ Hazard: prone to certain accidents
like cutting yourselves
○ If we think about elimination, we are telling
ourselves that we need to stop our workers in
performing a task that involves using utility 7. Biological safety cabinets
knives - HEPA filtration of air intake and exhaust
○ If we can't eliminate such hazards (using utility - Recirculates filtered air into laboratory
knives), the next thing we can do is look for - Ensure sterility
alternatives. 8. Fume hoods
■ Safe utility knives - No filtration of air
○ Engineering control - Exhausts chemical fumes outside the
■ Machines do the work (to avoid being laboratory
exposed to certain chemicals) - Suitable for chemicals and non-sterile work
○ Administrative Control - Never used for infectious agents
■ Trainings to ensure that the employee 9. Mechanical pipetting devices
has lesser chance of getting injured 10. Computer wrist/arm pads
○ PPE 11. Sensor-controlled sinks
■ Cut resistant gloves 12. foot/knee/elbow-controlled faucets
13. Eyewash station
Engineering Controls 14. Chemical Storage Cabinet
● Engineering controls remove the hazard from the - In terms of storage, corrosive and flammable
workplace create a barrier between the worker and the chemicals must be in small quantities as much
hazard i.e., installing physical barriers such as as possible
bullet-resistant enclosures 15. Safety showers
● Preferred among others because they make permanent - Installed where corrosive chemicals are being
changes that reduce exposures to hazards and do not used
rely on the worker’s behavior - Readily available to all personnel
Examples of Engineering Controls Administrative Controls

1. Puncture-resistant containers ● Are those that modify workers’ work schedules and tasks
2. Safety needles in ways that minimize their exposure to workplace
3. Biohazard bags hazards
○ Biohazard- refers to biological substances that ● Examples:
pose a threat to the health of living organisms ○ Developing a chemical hygiene plan
primarily that of humans ○ Developing SOP for chemical handling
2
○ Warning alarms ● Employers of health care workers must establish and
○ Labeling systems implement an infectious waste program
○ Trainings ● All biomedical waste should be placed in a bag marked
with the biohazard symbol and then placed into a
Work Practice Controls leak-proof container that is puncture-resistant and
● Practices that reduce the risk of exposure altering the equipped with a solid, tight-fitting lid. All containers must
way in which a task is performed to make it safe be clearly marked with the word biohazard or its symbol.
● Intended to reduce the likelihood of exposure by ● All sharp instruments, such as needles, blades, and
changing the way a task is being performed glass objects, should be placed into special
puncture-resistant containers before placing them inside
that bag and container
Laboratory Hazard Prevention Strategies
● All biomedical waste must then be disposed of, according
Work practice controls ● Hand washing after each patient to one of the recommended procedures
(general procedures or contact ● Highly pathogenic waste should undergo preliminary
policies that mandate ● Cleaning surfaces with disinfectants treatment on-site.
measures to reduce or ● Avoiding unnecessary use of ● Potentially biohazardous material, such as blood
eliminate exposure to needles and sharps and not products and contaminated laboratory waste, cannot be
hazard) recapping directly discarded. Contaminate noncombustible waste,
● Red bag waste disposal such as glassware, should be autoclaved before being
● Immunization for hepatitis discarded. Special attention should be given to the
● Job rotation to minimize repetitive discarding of syringes, needles, and broken glass that
tasks could also inflict accidental cuts or punctures.
● Orientation, training, and continuing Appropriate containers should be used for discarding
education these sharp objects.
● No eating, drinking, or smoking in
the laboratory FIRE HAZARD
● Warning signage ● Four factors causing fire: (Fire Tetrahedron)
○ Fuel
Personal Protective Equipment ○ Heat
○ Oxygen
1. Gloves
○ Uninhibited Reaction
2. Lab gown
■ The continuous reaction of fuel, heat,
3. Eyewash stations
and oxygen
4. Protective eyewear
● In order for fire to start, the four must be completed
5. Face shield
6. Face mask
7. Safety showers
8. Appropriate footwear
HAZARDS CLASSIFICATION: TYPES OF HAZARDS
Safety Hazards General Classification

1. Biological
2. Fire
3. Electrical
4. Chemical
5. Mechanical (physical)
6. Radiation
7. Compressed gasses
8. Cryogenic Materials
9. Ergonomic
BIOLOGICAL HAZARD
● Medical Waste - “may transmit infectious diseases”
○ Blood and body fluids, stool, urine, body
tissues, etc.
○ 3 types of disposal
■ Incineration
■ Chemical treatment
■ Autoclaving
● Discard sharps in puncture-resistant containers located
within the work area
● Needles should NOT be transported, recapped, bent,
or broken by hand
3
● The type of fire extinguisher hat can be used for all
classes is the Dry Powder
ELECTRICAL HAZARD
● Direct effect:
NFPA (National Fire Protection Agency) Hazard Label
○ Shock
○ Burns ● Each diamond represents different hazards
○ Death ● Flash Point
● Indirect effect: ○ Lowest temp. at which a liquid can give off
○ Explosion vapor to form an ignitable mixture in air near
○ Fire the surface of the liquid
● What to look out for to prevent hazards ○ The lower the flashpoint, the easier it is to ignite
○ Free cords
○ Removed grounding prongs sa plug
○ Any type of grounding shock being used
CHEMICAL HAZARD
● Employees must be notified of the potential health
hazards of the handled chemicals
○ Chemical storage equipment must be arranged
& labeled accordingly
○ Take note of storage requirements
○ MSDS (Material Safety Data Sheets)
■ Compiled information about the
chemicals being handled
■ Must be on file and available for every
chemical in the lab
■ Control measures in case of exposure
MECHANICAL HAZARD
● A.k.a Physical Hazards
● Hazards created by the use of or exposure to either
powered or annually operated equipment, machinery,
and plant
○ Centrifugation lapses
○ Lab glassware
RADIATION HAZARD
● Ionizing radiation can damage living tissue in the human
body
● Strips away electrons from atoms and break some
chemical bonds
● When these particles come in contact with organic
materials like the human tissue, it damages them causing
burns and cancer
● To reduce radiation exposure remember:
○ Distance
■ The farther from the source of
radiation, the lesser exposure
○ Shielding
■ Wearing pieces of equipment that
shield us from it
○ Time
■ The longer the exposure, the more
hazard
4
COMPRESSED GASES ● 10-14hrs: lipids and lipoproteins
● All compressed gases are hazardous because of the ● 48 hrs fasting- increase serum bilirubin
high pressures inside the cylinders. ○ Increase clearance of bilirubin which decreases
● Mixture of gases n a container having a certain pressure during fasting.
● We are putting pressure in the container ● 72 hrs fasting-increase triglycerides while glucose
○ Pressure: 40 psi at 21.1 degree celsius decreases in women to 45 mg/dL.
● All are hazardous because of the high pressure inside ● Basal state collection: glucose, cholesterol, triglyceride
the cylinders. and electrolytes
○ Can become certain missile Note: Basal state collection is early morning blood
collection, 12 hrs after the last ingestion of food.
● Requires fasting specimen: FBS, GTT, TAG, Lipid Profile
CRYOGENIC MATERIAL
test, gastrin and insulin.
● Liquid Nitrogen
● Cryogens DIET
○ Substances used to produce low very ● High protein diet-increase urea
temperature ○ Urea- breakdown products of proteins
■ Low as -153 degree celsius ○ Ammonia can be further converted into urea
● Ex: liquid nitrogen ● Glucose, lipids and catecholamines may show variation
○ Major hazard of liquid nitrogen are associated postabsorptive hormonal effects.
with the properties of extreme cold evaporation. ● High protein, low carbohydrate diets- increased ketones
in urine.
ERGONOMIC HAZARD ○ Ketones- comes breakdown of fats
● Factors in our environment that can harm our ○ High protein, low carbohydrate→ fats used as
musculoskeletal system energy
● Causes strain disorders ● Fat-rich food may increase potassium, ALP, TAG, and 5
● Primary contributing factors: HIAA
○ posture/ position ○ HIAA- 5-hydroxyindoleacetic acid
○ Applied force
■ Breakdown product of serotonin
○ Frequency of repetition
■ Serotonin- a hormone in the brain that
Prevention strategies
can affect our mood.
● Job rotation to minimize repetitive tasks (work practice
● Serotonin-rich food (banana, pineapple, tomato, and
controls)
avocado) increase the urinary excretion of 5-HIAA.
● Computer wrist/ arm pads (engineering controls)
● Caffeine increases concentrations of glucose; it promotes
● Placing comfortable seats
the release of catecholamines from the adrenal medulla
and brain tissue.
"Safety begins with the recognition of hazards and is achieved
○ Catecholamines- increases glucose,
through the application of:common sense,a safety-focused
decreasing release of insulin
attitude,good personal behaviour,good housekeeping in all
■ Flight and fight hormones.
laboratory work and storage areas,and above all, the continual
practice of good laboratory technique." ● Increased in obese person: LD, cortisol and glucose.
POSTURE AND POSITION

PATIENT PREPARATION
● Preferred position during phlebotomy: upright position
Pre-analytical Variables/ Factors contributing to the variation of
or supine
results:
● Changing from supine to sitting or standing position-
● Exercise
increased levels of albumin, enzymes, and calcium
● Fasting
○ Causes constriction of blood vessels and
● Diet
reduction of plasma volume
● Posture and Position
● Changing from sitting to supine- increase levels of
● Tourniquet application
proteins, lipids, BUN, iron, and calcium
● Tobacco Smoking
○ Since there will be a shift of water and
● Alcohol ingestion
electrolytes into your tissues, it causes
● Stress (anxiety)
hemoconcentration
● Drugs
● Changing from standing to supine position- decreased
levels of cholesterol, triglycerides, and lipoproteins
EXERCISE
○ Since this causes exovascular water to transfer
● Volume shifts between the vascular and interstitial
to your vascular systems and dilutes your non
compartments, volume loss by sweating and changes in
diffusible plasma constituents
hormone concentrations.
● Prolonged standing for more than 30 minutes- increased
● Transient increased in lactate, fatty acid, ammonia.
potassium
○ Lactate- produced when there is a lack of
○ due to the release of potassium to your
oxygen
muscles
○ Ammonia- waste product of your amino acids
● Prolonged bedrest- decreased plasma albumin
and proteins; product of deamination of ATP
○ Due to fluid retention
● Long-term increased in CPK, AST, LD and aldolase.
○ Enzymes found in skeletal muscle
Thus, it is advisable that we need to let patients rest for at least 15
● Increased in prolactin, testosterone and luteinizing
minutes to regulate and stabilize their bodily substances
hormone (LH)
● Elevated levels of proteins in urine (proteinuria)
TOURNIQUET APPLICATION
● Vigorous hand exercise (fist clenching) increases
● 1 minute application is recommended
potassium, lactate and phosphate.
● Prolonged application= hemoconcentration and
● Decreased plasma levels of FSH and LH in long distance
anaerobiosis
athletes.
○ Anaerobiosis - increase in the substances
○ FSH and LH- reproductive hormones
present in our body that does not need oxygen
● Increase levels: potassium, proteins (albumin), enzymes,
FASTING
lactate, cholesterol, and ammonia.
● 8-10 hrs: glucose
5
● Prolonged use of tourniquet with fist exercise- increase ○ Alkaline, phosphatase, cholesterol, and
potassium 1mmol/L phosphorus
● For measurement of lactate - tourniquet use should be ● Affected by gender (increased levels):
minimal and the patient should not clench his or her fist ○ Male: Albumin, ALP, creatinine, uric acid,
otherwise will result to elevated levels of lactate cholesterol, BUN
○ Female: HDL, iron, and cholesterol
TOBACCO SMOKING ● Affected by recent food ingestion:
● Increased in plasma catecholamines and cortisol ○ Increased levels: glucose, TAG, astrin, free
● Increased in glucose, growth hormone, cholesterol, calcium
triglycerides, ammonia, urea, lactate, insulin and urinary ○ Decreased levels: electrolytes (Cl-,K+,P+), ALP,
5- HIAA. AMS
● Increased plasma non esterified acid concentration
● Decreased plasma levels of vitamin B12 and elevated
thiocyanate
○ Tobacco has nicotine, which has an endocrine
effect on our body, thus elevating endocrine
hormones
ALCOHOL INGESTION
● Increased level of urate, triglycerides, and gamma
glutamyl transferase (GGT).
○ GGT= liver enzyme; test requested by the
physician since it is a serum marker for alcohol
related diseases
● Hypoglycemia (chronic alcoholism)
○ Alcohol consumption causes an increase in
insulin secretion which leads to lowering your
blood sugar level causing hypoglycemia
STRESS (ANXIETY)
● Affects adrenal hormone secretion
● Increased: catecholamines, cortisol, ACTH, prolactin,
insulin, albumin, glucose, and lactate
● Total cholesterol has been reported to increase with mild
stress, and HDL cholesterol to decrease by as much as
15%.
● Hyperventilation - affects acid-base balance
○ Conserving or needing of increased oxygen
● Since there is body tension, we begin to breathe a little
more shallow and lower our oxygen levels in the blood,
which sends signals to our brain that we are at stress.
DRUGS
● Hepatotoxic drugs can elevate liver function enzymes.
● Diuretics can cause decreased plasma sodium and
potassium.
○ Diuretics
■ also known as water pills
■ Common treatment for high blood
pressure
● Opiates cause increases in liver and pancreatic enzymes
○ Pang high na drugs
○ 1 content - Acetaminophen which has a direct
effect to our liver causing toxicity
PHYSIOLOGIC VARIATION
● changes that occur within the body such as cyclic
changes (diurnal or circadian) or those resulting from
exercise, diet, stress, gender, age, drugs, posture or
underlying medications.
● Affected by diurnal variation:
○ increased in AM: ACTH, aldosterone, cortisol,
and iron
○ decreased in PM: Acid phosphatase, Growth
hormone, Parathyroid hormone, Thyroid
stimulating hormone
- There is fluctuation in our body
- Most substances with diurnal variation are hormones
- That is why it is very tricky when we are ing requested to
collect samples for hormonal testing
- Timing is needed for collection Ex. ACTH - highest peak
is at 8AM
● Affected by age (increased levels):
6
LECTURE 2: EXPRESSING CONCENTRATIONS

PARTS 1-2
Prof. Fritdey Jad Doctolero, RMT, MLS(ALCPi)CM
August 23, 2021
DEFINITION OF TERMS
● Solution - a homogeneous mixture of two or more This formula is usually used when the problem asked for the
substances and can be in a form of solid, liquid, ot gas needed weight of a solute and the given is the percent
○ Solute - substance being dissolved concentration and either the weight or the total volume of the
○ Solvent - substance that dissolves desired solution :
● Concentration - how much of the solute is present per
unit of volume 𝑥 (𝑔)
𝑔 = 100 𝑚𝐿
[𝑥 (𝑚𝐿) 𝑑𝑒𝑠𝑖𝑟𝑒𝑑 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛]
2. %v/v
● Both solute and solvent are liquid
● Units: % v/v = mL per 100mL
This formula is used when the problem asked for the needed
volume of the solute given that the percent concentration and
desired solution is given in the problem:
Which among the 4 is very concentrated and the least 𝑥 (𝑚𝐿)

concentrated? 𝑚𝐿 = 100
● A = very concentrated (more solute)
● D = least concentrated/most diluted (less solute)
3. %w/w
How to express the concentration of solutions? ● Use the weight of the final solution rather than the
● Percent concentration volume
○ 0.9% Normal saline solution ● Units % w/w = grams per 100 g
● Molarity
○ 0.109 M buffered sodium citrate 𝑥 (𝑔)
● Molality 𝑔 = 100 𝑔
[𝑥 (𝑔) 𝑑𝑒𝑠𝑖𝑟𝑒𝑑 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛]
○ 5 moles/kg sucrose solution
● Normality
○ 2 N NaCl REMINDERS
○ 1 N = eq/L
● Having another way of solving given problems is okay as
Percent oncentration long as it arrives at the same answer
● Means per 100 or parts per 100 ● Take note of the units, make sure that before you
● Relationship substitute values, you always check the units
○ Solute/Solution ● Box your final answers every time
3 ways to express the concentration of a solution through % ● Do not round off answers in the middle of the solution,
concentration: always round off final answers to the hundredths place
1. % w/v (weight/volume) or two decimal places unless a specific instruction is
2. % w/w (weight/weight) given
3. % v/v (volume/volume) ● If the answer is not in two decimal places, it will suffice
● If whole number, hindi na lagyan ng two zeroes after
These 3 formulas are applicable when you want to determine the ● For solving and writing your answers, you can abbreviate
percent concentration especially in units of measurement bu make sure na
tama yung abbreviation na ginagamit
𝑤𝑒𝑖𝑔ℎ𝑡 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
𝑣𝑜𝑙𝑢𝑚𝑒
% = 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
𝑥100
What weight of glucose is needed to prepare 250 mL of a
𝑤𝑒𝑖𝑔ℎ𝑡 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 15% w/v solution?
𝑤𝑒𝑖𝑔ℎ𝑡
% = 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
𝑥100
𝑣𝑜𝑙𝑢𝑚𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 𝑥 (𝑔)

𝑥100 𝑔 = 100 𝑚𝐿
1. % w/v Solution:
15𝑔
𝑔 = 100 𝑚𝐿
𝑥 250 𝑚𝐿
● Used as a unit of measurement when the solute is a solid
and the solvent is a liquid 𝑔 = 37. 5 𝑔
● Grams of solute per 100mL of the total solution
● Example: 5% w/v
○ This means that there is 5grams of solute per ● It is asking for the needed volume of the solute
100mL of the total solution
● Units: %w/v = grams/100mL
1
● What you need to use is yung second na formula ng ● Preparation of the solution: Put 22.5 mL of ethanol in
w/v 127.5 mL of distilled water to make 150 mL of 15% v/v
● Substitute the values na given sa problem solution
● Remember when we say 15% w/v is the same as Countercheck
saying 15g/100mL times the volume of the desired ● Your solute and solvent should sum up to 150 mL
solution
PERCENT CONCENTRATION
A solution contains 24g of solute in 300mL solution. What What is the %w/w if 8.0 grams of copper is added to zinc to
is the percent concentration? produce 100 grams of an alloy?

𝑥100
𝑥100 solution:
solution: 𝑤𝑒𝑖𝑔ℎ𝑡 8.0 𝑔
% = 100 𝑥100
𝑤𝑒𝑖𝑔ℎ𝑡 24 𝑔
% = 300
𝑥100 = 8% w/w or 8 g/100 mL
8𝑔
=
100 𝑚𝐿
x100 or 8%
How would you make 200 mL of a 15% salt in a water
solution?
𝑥 (𝑔)
𝑔 = 100 𝑚𝐿
Describe how to prepare 200 mL of a 5% v/v solution of
methanol. solution:
15 𝑔
𝑔 = 100 𝑚𝐿
𝑥 200 𝑚𝐿
𝑥 (𝑚𝐿)
𝑚𝐿 = 100
[𝑥 (𝑚𝐿) 𝑑𝑒𝑠𝑖𝑟𝑒𝑑 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛] = 30 𝑔
Solution: Final answer: Place 30 grams of salt and add water up to
5 𝑚𝐿 200 mL.
𝑚𝐿 = 100 𝑚𝐿
x 200 mL
= 10 𝑚𝐿
Make 300 grams of a 20% w/w aqueous solution of sodium
10 mL of methanol is added to distilled water until 200 mL chloride.
final volume of the solution is prepared.
𝑥 (𝑔)
How much ethanol is needed to prepare 150 mL of 15% v/v
𝑔 = 100 𝑚𝐿
[𝑥 (𝑔) 𝑑𝑒𝑠𝑖𝑟𝑒𝑑 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛]
solution? How much dH2O is added in preparing the Solution:
solution? How to prepare the solution? 20 𝑔
𝑔 = 100 𝑔
𝑥 300 𝑔
𝑥 (𝑚𝐿)
= 60 𝑔
𝑚𝐿 = 100
Solution: Final answer: 60 g of NaCl is needed to make 300 g of a
15 𝑚𝐿 20% w/w solution.
𝑚𝐿 = 100 𝑚𝐿
x 150 mL
= 22. 5 𝑚𝐿 ethanol→ solutes volume
Volume of solution= volume of solute + volume of solvent

150 mL = 22.5 mL + volume of solvent
127.5 = volume of solvent which is dH20
Addressing the first question

● Use the second formula introduced for %V/V
○ Substitute the values
● If you forget about the final unit, apply cross units.
(pag mag cancel na)
● 22.5 mL volume of solute
Finding the volume of the solvent (second question)
● Use the formule:
○ Vol of soln = vol of solute + vol of solvent
● Substitute
● 150 mL = 22.5mL + vol. Solvent
● 127.5 = volume of solvent
Third question (How to prepare the solution?)
2
PART 2
Molarity 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒
𝐹: 𝑀𝑜𝑙𝑎𝑟𝑖𝑡𝑦 = 𝑀𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝐿𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
● MOLE
○ SI unit for the amount of a substance 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
○
23
1 M = 6. 02𝑥10 pieces
6 = 58𝑔/𝑚𝑜𝑙 𝑥 2 𝐿
■ Pieces - atoms, molecules or particles 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 = 6 𝑥 58 𝑥 2
23
■ 6. 02𝑥10 = Avogadro’s number = 696 grams
○ (mass of substance/MW)
○ Atomic weight (Atomic mass)
○ Seen beside the chemical symbol in the A solution contains 3.5 grams of hydrochloric acid in 1L.
periodic table How many mmol does it contain? (H: 1; Cl: 35)
○ ATOMIC/MOLECULAR WEIGHT
○ is the actual mass of the chemical particle
relative to the mass of the carbon atom 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑢𝑏𝑠𝑡𝑎𝑛𝑐𝑒
○ Sum of all atomic masses in a molecule 𝑚𝑜𝑙𝑒 = 𝑀𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡
Ex. What is the molecular weight of water?
1. Determine the atomic mass of oxygen and hydrogen 3.5𝑔
- O = 16 ; H = 1 (2) - bc water has 2 hydrogen = 36 𝑔/𝑚𝑜𝑙
- 16 + 2 _
- = 18 g/mol = 0. 0972 𝑚𝑜𝑙
- (H20 = 18 g/mol) _
1000𝑚𝑚𝑜𝑙
How many moles will 5 grams of water have? = 0. 0972 𝑚𝑜𝑙( 1 𝑚𝑜𝑙
) = 97. 33 𝑚𝑚𝑜𝑙
- (mass of substance/MW)
- 5/18
- 0.28 moles pr 0.28 mol ● Hindi involved ang formula for molarity
● The number of moles of solute in one (1) liter of solution ● Do no forget the cancellation of units
2 formulas: ● Yung number 2 may symbol sa taas which means na
yung number 2 ay repeating
𝑀𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑡 𝑥 𝑚𝑜𝑙𝑎𝑟𝑖𝑡𝑦 = 𝑔𝑟𝑎𝑚𝑠 / 𝑙𝑖𝑡𝑒𝑟 ● We need to convert to mmol because yan ang
required sa problem
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒 ● Para malaman mo kung saan ilagay yung 1 mol or 1
𝑀𝑜𝑙𝑎𝑟𝑖𝑡𝑦 = 𝑀𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝐿𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 mmol dapat alam mo yung required na units and yung
Unit: units na dapat icancel
● moles/L ● Since the two numbers are in the same level, you
● M multiply tthem both
● mol/L
There are 20g NaCl in 400mL of solution. What is its Make 300 mL of 6M NaCl
molarity? (Na: 23; Cl: 35)
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒

𝑀𝑜𝑙𝑎𝑟𝑖𝑡𝑦 = 𝑚𝑤 𝑥 𝐿 𝑜𝑓 𝑠𝑜𝑙𝑛
𝑀𝑜𝑙𝑎𝑟𝑖𝑡𝑦 = 𝑀𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝐿𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
20𝑔
6𝑀 = 58 𝑔/𝑚𝑜𝑙 𝑥 0.3 𝐿
= 58𝑔/𝑚𝑜𝑙 𝑥 0.4 𝐿
= 104. 4 𝑔
= 0.86 M 104.4 grams of NaCl is needed to make 300 mL of 6M NaCl or
104.4 grams is added to a solvent to make 300mL of 66M NaCl
● The final answer should be in a sentence form

How many grams of Sodium Hydroxide are contained in ● Remember liters of soln ang kailangan but the given is
500 milliliter of a 4 molar solution? (Na: 23 ; O: 16 ; H: 1) in mL so we convert it to liters

𝑀𝑜𝑙𝑎𝑟𝑖𝑡𝑦 = 𝑀𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝐿𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 Molality
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 ● The number of moles of solute in one thousand grams
4 𝑚𝑜𝑙/𝐿 = 40𝑔/𝑚𝑜𝑙 𝑥 0.5 𝐿 (1000 g) or 1 kg of solvent rather than the final solution
80g = weight of solute NaOH 𝑚𝑜𝑙𝑎𝑙𝑖𝑡𝑦 = 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝑘𝑖𝑙𝑜𝑔𝑟𝑎𝑚 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
Unit: moles/kg
If NaCl has a gMW of 58g/mole, how many of the solute is
added to 6 molar solution of NSS to come up with 2L? What is the molality of a solution if 127 grams of NaCl is
dissolved in 1000 grams of dH20?
3
= 58 grams of NaCl is needed to make 300g of a 2N NaCl
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒 solution
𝑚𝑜𝑙𝑎𝑙𝑖𝑡𝑦 = 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝑘𝑖𝑙𝑜𝑔𝑟𝑎𝑚 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
Calculate the barium chloride concentration in mEq/L of a

= 2. 19 𝑚𝑜𝑙𝑒/𝑘𝑔 solution prepare by diluting 25 grams of barium chloride
2L.
How many grams of NaCl would be in a 100g of a 1 molal

solution? ● Barium = 137.33; Chloride = 35.45
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒 𝑚𝑤 137.33 + 35.45 (2)

𝑚𝑜𝑙𝑎𝑙𝑖𝑡𝑦 = 𝐸𝑤 = 𝑣𝑎𝑙𝑒𝑛𝑐𝑒
= 2
𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝑘𝑖𝑙𝑜𝑔𝑟𝑎𝑚 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
1 𝑒𝑞
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 = 104. 115𝑔/𝐸𝑞 ( 100 𝑚𝐸𝑞 )
1 𝑚𝑜𝑙𝑎𝑙 = 5.8𝑔/ 𝑚𝑜𝑙 𝑥 01 𝑘𝑔 𝑔
= 0.104115 𝑚𝐸𝑞
5. 8 g = weight of Nacl 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒
𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦 = 𝑒𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝑙𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
Normality 25𝑔
= 0.104115 𝑔/ 𝑚𝐸𝑞 𝑥 2𝐿
● Number of equivalent weights per Liter of solution
𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦 = 𝑒𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝑙𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 𝑚𝐸𝑞
= 120.06 𝐿
● Equivalent Weight
○ The mass of an element or compound that will
combine with or replace one mole of hydrogen How many milliequivalents are in 1L of a 4.5 N HCl
○ Is dependent on the total charge of the positive solution?
ion, or the valence, of the element
○ UNIT: g/ eq
𝑀𝑊 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒
𝐸𝑊 = 𝑉𝑎𝑙𝑒𝑛𝑐𝑒 𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦 = 𝑒𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 𝑙𝑖𝑡𝑒𝑟 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
● If the compound is an acid, an equivalent is the quantity 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒
of substance that contains one replaceable hydrogen. 4. 5 𝑁 = 𝑔
36.45 𝑥 1𝐿
● If it is a base or a salt, an equivalent is the quantity of a 𝐸𝑞
substance that will react with one replaceable hydrogen. 164. 025𝑔 = 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝐻𝐶𝐿
○ Tingnan lang ang number ng H
○ Ex: sulfuric acid H2SO4 Another formula to get the equivalence:
■ Valence: 2 𝑔𝑟𝑎𝑚𝑠 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒
𝐸𝑞 = 𝐸𝑤
NORMALITY 164.025 𝑔
Valence EW = 36.45 𝑔/𝐸𝑞
NaCl 1 58.5 = 4.5 Eq
BaCl2 2 104.12
K2CO3 2 69 convert:
1000 𝑚𝐸𝑞
CaSO4 2 68 𝑚𝐸𝑞 = 4. 5 𝐸𝑞 ( 1 𝐸𝑞
)
AlCl3 3 44 = 4500 mEq
Fe2(SO4)3 6 66.6
What is the normality of a solution that contains 150 grams

of sodium chloride per liter?

150 𝑔
= 58 𝑔/ 𝑒𝑞 𝑥 1𝐿
= 2.59 N or 2.59 Eq/L
How do you make 500 mL of a 2N NaCl?

2 𝑒𝑞 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒
𝐿
= 58 𝑔/ 𝑒𝑞 𝑥 0.5𝐿
= 58g
4
LECTURE 3: CONVERSIONS
Prof. JC Louise Bandala, RMT
August 23, 2021
CONVERSIONS
● Molarity to normality
● Normality to molarity Convert 3N H2SO4 to a percentage (MW: 98)
● Molarity or normality to %w/v
● mg/dL to mmol/L or vice versa 𝑒𝑞 𝐿 𝑚𝑜𝑙𝑒 𝑔
𝑥 𝑥 𝑥 𝑥100
● mg/dL to mEq/L 𝐿 1000𝑚𝐿 2𝑒𝑞 𝑚𝑜𝑙𝑒
Molarity to Normality and Normality to Molarity N = eq/L % = g/100 mL valence = eq/mole mw= g/mol
● N = (M)(valence)
● M = N / valence How would we be able to get g/100 mL from eq/L?
Valence - easy to know if hydrogen and hydroxide is present 3𝑒𝑞 𝐿 𝑚𝑜𝑙𝑒 98𝑔
3𝑁 = 𝐿
𝑥 1000𝑚𝐿
𝑥 2𝑒𝑞
𝑥 𝑚𝑜𝑙𝑒
Convert 6N NaOH to molarity ● When we compute for normality, kailangan ang valence =
eq/mole
𝑁 ● Cancel units
𝑀= 𝑣𝑎𝑙𝑒𝑛𝑐𝑒
6𝑁 98 𝑥 3 294 𝑔 0.147 𝑔 100
𝑀 = 1 1000 𝑥 2
= 2000 𝑚𝐿
= 𝑚𝐿
𝑥 100
=
= 6𝑀 14. 7% 𝑤/𝑣 𝐻2𝑆𝑂4
● We already have the 294/2000
● Remember mL ni siya dili na siya 100
● Multiply both numerator and denominator by 100
Convert 10M H2SO4 to normality
𝑁 = 𝑀 𝑥 𝑣𝑎𝑙𝑒𝑛𝑐𝑒 CONVERSIONS:
● mg/ dl to mEq/L
= 10𝑀 𝑥 2 ● And vice versa
= 20𝑁
A sodium concentration is reported as 250 mg/dL. What is
Conversions its concentration in mEq/L? (MW = 22.99)
● % to N or M and vice versa
● Recall that when we say percent concentration that is N= ?
equivalent to grams per 100mL Valence = 1 eq/ mole
Example: 250 𝑚𝑔 10 𝑑𝐿 𝑔 𝑚𝑜𝑙𝑒 1𝑒𝑞 1000 𝑚𝑒𝑞

𝑑𝐿
𝑥 𝐿
𝑥 1000 𝑚𝑔
𝑥 22.99 𝑔
𝑥 1 𝑒𝑞
250 𝑥 10 𝑥 1000
Convert 30% NaCl to molarity (MW: 58.44) 1000 𝑥 22.99
= 108. 74 𝑚𝐸𝑞/𝐿
% = g/100mL 𝑚𝑔 𝑑𝐿 𝑔 𝑚𝑜𝑙𝑒 𝑒𝑞 𝑚𝑒𝑞

Molarity = moles/L 𝑑𝐿
𝑥 𝐿
𝑥 𝑚𝑔
𝑥 𝑔
𝑥 𝑒𝑞
= 𝑚𝐸𝑞/𝐿
MW = g/mole
30𝑔 1000𝑚𝐿 𝑚𝑜𝑙𝑒
100𝑚𝐿
𝑥 𝐿
𝑥 58.44
= 5.13M NaCl A sodium concentration is reported as 250mg/dL. What is
its concentration in mEq/L? (MW = 22.99)
Valence = eq/mole
MW = g/mole
Convert 30% NaCl to molarity (MW: 58.44)
10 𝑚𝑔 10 𝑑𝐿 𝑔 𝑚𝑜𝑙𝑒 1000 𝑚𝑚𝑜𝑙
% = g/100mL 𝑑𝐿
𝑥 𝐿
𝑥 1000 𝑚𝑔
𝑥 40.08 𝑔
𝑥 1 𝑚𝑜𝑙
Molarity = moles/L
MW = g/mole = 2.495 mmol/L
30𝑔 1000𝑚𝐿 𝑚𝑜𝑙𝑒
100𝑚𝐿
𝑥 𝐿
𝑥 58.44
= 5.13M NaCl
1
A calcium concentration is reported as 10 mg/dL. What is
its concentration in mmol?liter? (MW = 40.08) g/mol
10 𝑚𝑔 10 𝑑𝐿 𝑔 𝑚𝑜𝑙𝑒 1000 𝑚𝑚𝑜𝑙

𝑑𝐿
𝑥 𝐿
𝑥 1000 𝑚𝑔
𝑥 40.08 𝑔
𝑥 1 𝑚𝑜𝑙
= 2.495 mmol/L
Summary
● When converting, be mindful of the units
● Remember how to compute Normality and Molarity
because its units will help you convert one to the other
2
3
4
LECTURE 4: OPTICAL TECHNIQUES

Prof. Bernardo Ordaneeza Jr., RMT
August 31, 2021
PART ONE
■ wavelengths = how far away the
ELECTROMAGNETIC RADIATION (EMR) crests and the troughs of each wave
● Different types of electromagnetic:
○ Radio waves = longest
○ Microwaves
■ used in satellite broadcasting
■ Cellphones signals = bordering
between radio and microwaves
■ Microwave oven
○ Infrared
■ Remote controls
■ Being radiated by heat
■ Heat sensitive cameras
○ Visible spectra
■ Sensitive sa atong eyes
○ Ultraviolet - beyond the visible spectra
○ X-rays
○ Gamma Rays/ cosmic rays - most powerful
● Can be described as discrete packets of energy

(photons) or waves
● Sinusoidal oscillation of both electrical and magnetic
fields at right angles to each other and to the direction of
propagation
● Main principle of spectrophotometry involves light
● Light is a form of EMR
○ Oscillation = wave
○ Disturbance in the electrical and magnetic field
■ Field in physics is everywhere
■ Whenever there is a disturbance in
that field, then electrons will exist
■ According to physics, whenever there
is a disturbance in the electrical field,
there is always a corresponding
disturbance in the magnetic field at
right angles to each other
● Remember: it is not necessary that it’s automatically
parallel to the Y axis and the magnetic field is
automatically parallel to the X axis
● Often times, the oscillation comes at the right angle or at
a certain angle compared to the Y axis and X axis ● We have different parameters or dimensions of a wave
● MAIN TAKE AWAY: ● Electromagnetic radiation is just an escalation (?) of the
○ Electromagnetic radiation is an oscillation/ electromagnetic field
wave of both electrical and magnetic fields at ○ It has 3-dimensional structure
right angles to each other ● Wavelength - peak to peak
○ Can also be from valley to valley
○ Just arbitrary because we can flip this around
○ Waves do not really perfectly orient themselves
to the X or Y axis
○ Peak to peak of the oscillation or from one point
until it returns to that same point is called
wavelength
○ Symbolized by the greek letter lambda (λ)
○ One cycle = one wavelength
● Amplitude - from midline to its peak (how high or how
deep the valley is
● FM or AM (Radio)
○ AM - amplitude modulation
○ Because the way you switch the stations, the
different radio stations will broadcast at the
● Spectrum same frequency but different amplitude
○ Gradient
○ A family of electromagnetic radiation of varying
wavelengths
1
𝑓𝑟𝑒𝑞𝑢𝑒𝑛𝑐𝑦 (𝜈) = 𝐻𝑧 𝑜𝑟 𝑐𝑦𝑐𝑙𝑒/s ● If you see an equation where one element is something
● Another dimension of the electromagnetic radiation that is equal to something that is constant divided by the
● Symbolized by nu = ν wavelength, if it’s in the denominator, it is inversely
○ Pertains to number proportional
○ Cycles per sec ● That’s why decreasing the frequency, increases the
● Unit for frequency is Herts (Hz) wavelength
● Frequency - no. of oscillations in one second ● The shorter the frequency, the longer the wavelength
● Ex. propagates in 5 seconds ○ In reality, it’s not the case
● How to determine frequency? ○ C = speed of light = vacuum
○ How many cycles in one second? ○ A vacuum is a space where there are no
○ How many cycles was produced by the wave in particles in it
one second? ● What if light encounters a material like glass?
● 1 Hz or 1 cycle/ second ○ The speed decreases
○ Because particular electromagnetic energy
holds particular energy, so it could not really
change the frequency
○ Because the photon or the light holds an
energy that cannot be created nor destroyed
Summary:
● For particular electromagnetic radiation, there is a
corresponding energy
● The higher the frequency, the higher the energy
𝑠 1 ● That energy cannot be created nor destroyed. It could
Period (p) = 𝑐𝑦𝑐𝑙𝑒
= 𝑣 just transform into other forms of energy
● Frequency = speed of light/ wavelength which means the
● period - inverse of frequency
frequency is inversely proportional to the wavelength
● Periodicity - seconds/ cycle
● When electromagnetic radiation reaches a material, it
● How many seconds for a cycle to occur?
doesn’t change its energy
● Frequency remains constant
What will happen if we have electromagnetic radiation with a
● Electromagnetic radiation changes its speed, it slows
shorter wavelength?
down when it encounters a material
● When it changes its speed, let’s say it slows down, but
the frequency remains the same, how do you
compensate for the speed?
○ The wavelength increases to compensate for
the change of light
Important things to know:

● Still propagates the same speed (5s) ● Wavelength
● The behavior of the wave becomes more active ● Frequency, how many cycles per second?
○ Due to its shorter wavelength ● Periodicity- reciprocal of the frequency
● Shorter wavelength = higher frequency ○ How many seconds will it take for one cycle to
○ Shorten the wavelength by 2 fold, the frequency occur
will also increase by 2 fold
○ From 1 hz it becomes 2 cycle/sec (2hz)
● The energy of electromagnetic radiation is directly
proportional to the frequency
○ The higher the frequency, the more energetic
the electromagnetic radiation is (which shows)
○ Oscillation is much more active
■ Still travels the same way and speed,
but the frequency changed due to
higher energy
● It means that it would take ½ second for a cycle to occur

● The same is true for a wavelength with a frequency of
4hz, the periodicity is ¼
Radiofrequency
● Decrease the wavelength
● The waves will still travel at the same speed
● It will still take 5 seconds for it too reach one end to the
other
● There is more oscillation
● The shorter the wavelength, the higher the frequency
● Application
Relationship between energy and frequency ○ Broadcasting
● Directly proportional ○ Cellphone signal
● Represented by ○ Television
○ E= hv ○ Walkie talkies
● Very wide wavelength
𝑐 (𝑠𝑝𝑒𝑒𝑑 𝑜𝑓 𝑙𝑖𝑔ℎ𝑡) ○ 1 feet to the other would take 100,00 km to 1
Frequency (v) = meter
λ (𝑤𝑎𝑣𝑒𝑙𝑒𝑛𝑔𝑡ℎ)
● 1-meter wavelength of electromagnetic radiation o
2
100,000km, they are all under radiofrequency Visible Light (Blue)
λ v λ v
100,000 km 3 Hz 490 nm 610 THz
1m 300 mHz 450 nm 670 THz
● 3 Hz means 3 cycles per second
● Mega is 10^6 = 300,000,000 cycles per second
Visible Light (violet)
● Our eyes are not sensitive to radio frequencies
● Radiofrequency could travel large distances the reason
why they are used in radio broadcasting ● More energetic than your red
λ v
Microwave 450 nm 670 THz
400 nm 750 THz
● Wavelengths ranging from 1mm to 1m

● Frequency is 300 MHz to 300GHz
● Giga is 10^9 = 300,000,000,000
● More energetic than radio frequencies
● We can’t see microwaves
λ v
1 km 300 MHz
1 mm 300 GHz
Infrared
● Different textbooks will give different wavelengths
● 1 mm--10 μm wavelength
● Infra= below
● Infrared= below red
λ v
1 mm 300 GHz
10 μm 300 THz
● THz is 10^12 = quadrillion
● 30 quadrillion cycles per second because it’s very short
● Relatively more energetic then radiofrequency and
mircowave
Visible Light (Red)

● No clear cut boundary between colors because it is
subjective
● Eyes are sensitive ● Infrared is being radiated by heat
● No color in reality ○ Ex. cameras with thermometers
λ v ○ Expressed in nanometers (10^-9)
700nm 430 THz
635nm 480 THz
Visible Light (Orange)
λ v
635 nm 480 THz
590 nm 510 THz
Visible Light (Yellow)
λ v
590 nm 510 THz
560 nm 540 THz
Visible Light (Green)
λ v
560 nm 540 THz
520 nm 580 THz
3
● The only visible range is from red-violet. Anything that ● When you change the wavelength, there is a change in
goes beyond his range is invisible to our eyes the color or in the type of electromagnetic radiation
●
Ultraviolet
● Insects can be sensitive to UV light. Ex. butterflies.
○ Flowers reflect UV light
● Causes mutation that could lead to cancer
● Ionizing radiation
○ very energetic
● Visible light to low radiofrequency: non-ionizing
○ Thus, mutations due to being near to a cell
tower or having cellphones in your pocket is a
myth ● If you go for the intermedite, its yellow
λ v
100 nm 3 PHz
10 nm 30 PHz
● Longer wavelenth - red
X-Rays ● The longer the wavelength, the lower the frequency
● Gets more energetic than ultraviolet
● The limit for radiography: twice a year
● Can cause mutations
● Hexa - 10 to the power of 18
● it is so energetic that it can penetrate flesh
○ The reason why we use x-rays to have image
on the human body, although bones are
opaque
○ Flesh is transparent
● Wavelength is ranging to 1 nm to 10 picometer ( ten to
negative 12)
● If you combine the different lenghts in a spectra, you’ll
see white
● White light
○ combination of different wavelengths of visible
spectra
○ Polychromatic - many colors
λ v
1 nm 300 PHz
10 nm 30 EHz
Gamma Rays
● Most energetic
● Produced by radioactive materials, neutrons stars,
pulsars, quasars, and black holes
○ Because the universe is littered with energetic
stars, we are actually experiencing a ring of
gamma rays right now General Rule:
○ So energetic, it could penetrate the whole
planet
λ v
10 pm 30 EHz
1 pm 300 EHz
Electromagntic Spectrum
● What your eyes perceive, the thing absorbs

● When you see a red apple, does it mean that the apple is
red?
○ No, because it just reflects the red light
In the visible spectra, there is a change in color ○ If you shine it with a white light (which is
● If you shorten the wavelength, its violet composed of different colors of visible spectra),
the apple absorbs all the wavelengths of visible
spectra except red
4
○ That's why you are seeing red (being reflected)
● In a test tube, you added the serum and reagent
○ the color reaction that is apparent after reacting
the serum with the reagent is red
○ In spectrophotometer, we’re going to choose
the color or the wavelength that the particular
solution absorbs, and not the one reflected
○ If red, the best wavelength that we use for
spectrophotometer is green
● In spectrophotometry, what were after is not the light that
is reflected by the solution, but the light that is absorbed
by the solution
● If the solution is red, the wavelength that is being
absorbed is green ● Light bends at different corners.
● If colorless, it uses a different wavelength ● Light bending of light as it passes around the end of an
object.
Summary: ● Amount of bending depends on the relative size of the
● The visible spectra is composed of electromagnetic wavelength of light to the size of the opening .
radiation with varying wavelengths, energy, and ● If the opening is much larger than the lights wavelength,
frequency the bending will be almost unnoticeable.
○ different wavelength corresponds to different ● Slit
color ○ If we have a wide opening in relation to its
wavelength, there is a bending of the wave but
● Wavelength of interest we are going to use is the one the bending is much narrower.
being absorbed by the solution. ● The smaller the slit, the bending becomes more
Constructive Interference noticeable; bends more.
● property of the wave to bend around corners or slit.
waves are in-phase

● They align
● They travel together and they collide but in collision, the
peaks and the troughs are aligned.
● Points where it coincide with each other, once it reaches
the screen it is much brighter
○ Why? -- constructive interference
■ Waves coming from slit 1 and 2
coincide.
● Peaks where it coincide with the troughs of the 2 slits
○ They become dark.
○ Destructive interference.
● Interference pattern = Bright, dark, bright, dark
○ Combination of diffraction interference.
● The wave constructs itself.
● Amplitude of your wave increases because it reenforces.
Destructive Interference
waves are out-of-phase

● Out of phase ● Bending of diffraction depends on the
○ They collide, the peaks aligned with the troughs wavelength.
- it destroys itself. ○ The longer the wavelength the
more it bends
● Using diffraction grading
○ RED bends more
○ Purple bends less
Pic left: true interference pattern of white light.

● White light - combination of all the colors.
Diffraction
5
PART TWO
light is low, then the computer in the
SPECTROPHOTOMETRY spectrophotometer will translate that to high
● Spectrophotometry = principle concentration
○ Principle = how it works, why it works?
● Spectrophotometer = Instrument
● Measurement of the light transmitted by a solution to
determine the concentration of the light-absorbing
substance in the solution
● If the concentration of the solution is low, and when u

shine a high intensity green light, gamay lang na green
light ang ma absorb. Thus, the transmitted light is
relatively high.
● So, the photon detector detects that there is a high
● Cuvette - sample/ reagent holder used in intensity of transmitted light. Thus, the commuter will
spectrophotometer translate it to low concentration.
● Incoming light = incident light Basic Components:
● Outgoing light = transmitted light ● Light source
● Spectrophotometry is the measurement of this ● Entrance slit
transmitted light coming from this cuvette ● Monochromator
● Concentration = main reason why we do clinical ○ White light is a polychromatic light
chemistry ○ Polychromatic - composed of many
○ To know the concentration of a given analyte colors/different wavelengths
● Exit slit
● Sample holder (cuvette)
● Photodetector
○ Detects the transmitted light at the other side of
cuvette
● Read-out device
● All spectrophotometer have these basic components
● Glucose with Dinitrosalicylic acid (DNS)

● If u want to know the concentration of glucose in a given
serum or plasma, u must add a reagent (in this case
DNS)
● It gives off a color reaction. In this case, the color is red.
So, we use the green wavelength in spectrophotometer
● But before that, take a look at the 5 cuvettes, which do
you think has the highest amount of glucose in it?
○ The darker the color, the higher the
concentration of a substance
○ Lighter color - lover concentration of glucose
● Colorimetry - use our eyes and compare it with a
standard; eyes are subjective
● Spectrophotometer = instrument that measures
objectively the intensity of the color
○ It makes use of a light to determine the ● Single beam spectrophotometer
concentration of the light absorbing substance ● Monochromator = diffraction grating
in a solution ● Ex. (high concentration) 5% is being read by
● Ex. if a substance contains high concentration and once photodetector and is interpreted by the
u shine green light (high intensity), it absorbs the green spectrophotometer as high concentration
light, and because it is higher concentration, much of the ● The lower the transmitted light, the higher the
green light is being absorbed, And what is being concentration.
transmitted is gamay nalang na light.
○ Photodetector at the end

■ Detects the photons
■ Detects the intensity of light that is ● If the concentration is low, and has 95% transmitted light,
transmitted the spectrophotometer will interpret that one as low
○ If the photodetector detects that the transmitted concentration.
6
● We could not really measure directly the amount of light ● Temperature
that is absorbed by the sample ○ Depending on the type or source of radiant
○ We cant put an instrument inside the sample production, temperature might be a problem
because: ○ Especially if its a metal filament heat up may
■ It could interfere with the reaction of affect testing process
■ Impractical
● Incident light - should be constant Spectrophotometer
○ Light source should give the same intensity of ● Big instrument, has a robotic arm that mixes the samples
light at all times and the reagent, which puts it in the cuvette and into the
● Absorbed light - absorbed in the sample spectrophotometer
○ If the absorbed light is low, then the transmitted ● One thing that you must remember, in all of these
light is high and if the absorbed light is high, spectrophotometers it has same components
then the transmitted light is low ○ Those components are aligned in the same
○ We indirectly measure this light by measuring way
the transmitted light ○ Light source → entrance slit → monochromator
● Transmitted light → exit slit → cuvette → photo detector →
read-out device
LIGHT SOURCE ● When it comes to temperature, there are certain smaller
(Radiant Energy Source, Henry) spectrophotometers where the light source heats up
● Provides the energy (in the form of light) that the sample which affects the overall environment inside that
will modify or attenuate by absorption spectrophotometer
● The light produced is polychromatic ○ Chemical reactions are dependent on
○ I.e., visible wavelengths are present in one temperature
white light ○ You have to pick a light source that would not
● Must be powered by a regulated power supply give off that much heat
○ If no regulator, any fluctuation in the electricity ● Spectrophotometers in the laboratory (airconditioned) to
will result in the flickering of the light which is regulate the outside temperature and avoid it from
not good overheating
○ Constant and steady stream of light (must have ○ Aircon inside the lab is for the instruments
steady voltage)
● 2 types of Light Source (according to the light produced
Light Source (Radiant Energy Source)
by that light source)
1. Continuum Source
2. Line Source
→ difference is the characteristic of light being produced
by that light source
● Spectrum of visible light

● From far ultraviolet to near infrared
● Continuum source - much of the light given
peaks sa blue (doesn't mean it gives off blue
light, it also gives off other colors) ● Incandescent tungsten or tungsten-iodide lamp
○ Continuum source of colors ○ ~15% visible, the rest near IR (Bishop)
○ X axis = intensity ○ Typical clear bulb with a filament inside
○ Intensity of the light that is given off ● Tungsten or tungsten-halogen lamp
by this light source slowly decreases ○ Visible region in the EMS (Henry)
as a function of the wavelength ○ Follow kay henry
● Line source - much more limited range of colors ● Deuterium discharge lamp
that it can give off ○ Routinely used to provide UV Radiation
○ Ex. it gives off near infrared (continuous emission down to 165 nm)
● Continuum source is wider, line source is ○ Hydrogen with an extra neutron
narrower (spectrum of light that it can give off) ○ Heavier hydrogen
● A continuum source emits radiation that ● Hydrogen lamp
changes in intensity very slowly as a function of ○ UV radiation
wavelength. ● Xenon discharge lamp
● A line source emits a limited number of discrete ○ UV and visible range
lines of radiation which has limited range of ○ Noble gas
wavelength ● Mercury arc lamps or low-pressure mercury and sodium
vapor lamps
Factors in choosing a light source: ○ Sharp lines in the UV and visible regions
○ Routinely used to provide UV radiation
● Range
○ Low-pressure pertains to the pressure of the
○ Range of wavelengths that it could give off
gas inside the bulb
○ Range of colors the light source is capable of
○ It doesn’t mean na pag line source isa lang, it
giving off
could also give off different wavelengths of light
● Spectral distribution within the range
○ Used in fluorometry
○ What is the intensity of the different
○ Yellowish in color since sodium gives off a
wavelengths within that range
yellow color
● Source of radiant production
● Medium and high-pressure mercury lamps
○ Source of production of light (is it metal or
○ Continuum form UV to the mid-visible region
excitation of halogen gas inside (neon light)?)
● Hollow cathode lamp
○ Why does it light?
○ Use in AAS
● Stability of the radiant energy
○ AAS- Atomic Absorption
○ How stable and consistent the light source
Spectrophotometry
gives off a particular energy?
7
● Nernst glower nd Globar (SiC) violet
○ IR ○ In that way, we are dissecting the white light
○ Nernst- electrically heated rod off rare earth into different monochromatic light
element oxides ● Nominal wavelengths
○ Globar- uses silicon carbide is heated up ○ The wavelength (in nm) at peak transmittance
1200°C ○ Wavelength at the highest level
○ Heat emits infrared ● Spectral bandwidths
○ Range of wavelengths above one-half peak
transmittance
○ Half-powerpoint
○ Full width at half peak maximum
○ Range of wavelengths above ½ peak
transmittance
● Bandpass
○ Total range of wavelengths transmitted
○ Much wider
Entrance and Exit Slits

● Entrance Slit
○ Reduces stray light
○ Prevents scattered light from entering the
monochromator
○ Because it gives off noise
○ As much as possible, the light passing from the
light source to the photodetector must be
consistent
○ If you have noise, there might be problems in
your read-out
○ Allow a single beam of polychromatic light to
enter the monochromator
○ Related to the reason why spectrophotometerry ● A - Peak transmittance
components must be in the dark ● The narrower the spectral bandwidth and the
higher the peak transmittance, the better
Bandpass or Bandpass width

● Range of wavelength
● determines the efficiency of monochromator
● generally, the narrower the range, the more desirable
● prisms & gratings <5 nm
● interference filters = 10 to 20 nm
TYPES OF MONOCHROMATOR
● Prism
● Exit Slit ● Diffraction Gratings
○ Allows only a narrow beam of the spectrum to ● Filters
pass through the cuvette
○ Narrows down the light coming from the a. Prism
monochromator ● A wedge-shaped piece of glass, quartz, or NaCl
○ Choosing the wavelength of light that passes ● a narrow beam of light is refracted as it enters
through the sample by adjusting the exit slit up the prism (more dense material)
and down ● can be rotated, allowing the desired wavelength
○ If you bring it down, the red light becomes to pass through an exit slit
yellow or orange light or rotate the
monochromator
Monochromator
● A device that produces light of specific wavelengths from
a light source
● Produces monochromatic light
○ Light radiation of a single wavelength
○ Choose a single wavelength of light to measure
the light-absorbing substance in the cuvette
● Prism is the simplest monochromator

● In refraction, the red disperse at an angle lower than the
8
and allow only a limited domain of this
spectrum to pass through to the sample
II. Interference Filters

● simple, least expensive
● Semi-transparent
○ 50% passes through, 50% is reflected
● made by placing semi-transparent silver films
● If you adjust the exit slit, you should also adjust the on both sides of a dielectric field such as MgFl2
cuvette ● produce monochromatic light based on the
principle of constructive interference; eliminates
b. Diffraction Gratings other wavelengths by destructive interference
● most commonly used monochromator ● wavelength depends on the thickness of the
● better resolution than the prism gap between films
● made by cutting parallel grooves or slits into an
aluminized surface of a flat piece of crown
glass
● wavelengths are bent as they pass a sharp
corner
● Cloud have slits or just a reflected material;
essentially the same
● The polychromatic light is coming from this direction, but

our goal is the monochromatic light (red light)
○ We adjust the layers so that it is conducive for
constructive interference of red light
○ Once the red light enters, half of it is reflected,
half of it is scattered
○ Until such time that the red light has a higher
amplitude or being reinforced or more intense
in the other side of the interference filter
● For blue wavelengths, once it is reflected back, it is

disruptively interfere
○ So it will be eliminated as long as other colors
○ Less intense than the red light
● Filters have a higher bandpass than prisms and gratings
because of this path that some of the wavelengths might
leak (semi-transparent silver layer)
● Principle of IF: some wavelengths are constructively
c. Filters interfere while other disruptively interfere because the
● isolate monochromatic light, or at least as way the light interacts between the two semi-transparent
narrow a range of wavelengths of light as silver layer
possible, and direct it to the sample or to the ● Out of phase: disrupt, in phase: construct
photodetector
● Easiest way
● two types:
○ absorption filters
○ interference filters
I. Absorption Filters
● Ex. parol
● absorb regions of the electromagnetic spectrum
9
■ If the path length is wider, the higher
the light-absorbing substance will be
CUVETTES hit by the incident light
■ Length of the cuvette doesn't change
because it is solid
■ 10 cm cuvette pathway - sensitive
● Even small amounts of
concentrations of light
absorbing substance will be
detected by the
spectrophotometer
Types of Cuvettes
● fused silica or quartz
○ for UV (𝜆 < 350 nm)
○ Could still be used for visible light, but ideal for
UV light
● analytical cell or sample holder

● is used to hold the solution in the instrument whose
concentration is to be measure
● must be made of material that is transparent to radiation
in the spectral region of interest
● factors considered
○ ideal shape
● silicate glass (alumina silica and borosilicate)
■ Oftentimes square, but may be
○ 350 nm < 𝜆 < 2000 nm (visible region)
circular (not ideal) or rectangular
○ Visible spectra, UV light, par infrared region
■ The other two sides is transparent
○ Borosilicate glass for specrophmety analysis of
while the other two side is frosted
alkaline solutions
● Frosted side- part of the
○ For acidic, use tough glass
cuvette that you are going to
● Plastic
handle using your hands
● If you are going to handle it
on its transparent side, you
may leave fingerprints that
may give noise to your
analysis using the
spectrophotometer
■ Why square and rectangle, but not
circle?
● Circle - when you shine a
light, there would be
different angles that the light
travels
➢ this would cause a
problem since there ○ Most commonly used
may be an interaction ○ visible and UV range
between a light and ○ can present problems related to tolerances,
the glass that could cleaning, etching by solvents, and temperature
give off different deformations
readings ○ especially used for automated analyzers
● If square, the lights travels because they are disposable
in one direction ○ problem: once you clean it, you must be careful
■ Square cuvettes have plain parallel not to put scratches on it
optical surfaces and a constant light ■ since spectrophotometer is sensitive
● They have an advantage ■ Any scratch could lead to different
over round cuvettes that refraction of light that could give noise
there’s less error in the lens to your reading
effect , orientation in the
spectrophotometer, and Cuvettes
refraction
● must be clean and optically clear
● If circular cuvettes, you can
○ because etching or deposits on the surface
orient it in any orientation
affect absorbance values
while in square cuvette, you
○ scratched optical surfaces scatter light and
can orient it in one way or
should be discarded
another
● Cuvettes used for measurements in the UV region should
○ path length
be handled with special care
■ Generally, cuvettes are small (1 cm in
○ Especially fused silica or quartz
size)
● How to clean?
■ The smaller the path length, the better
○ Use tap water or distilled water (much better)
■ The amount of light-absorbing
○ Alkaline solutions should not be used because
substance that is being subjected to
it might dissolve the glass
incident light depends on the path
○ May be cleaned in a mild detergent or soap in a
length
mixture of concentrated hydrogen chloride to
10
water to ethanol in a ratio of 1:4
PHOTODETECTORS
● Converts transmitted light energy into an equivalent

amount of electrical energy
● We measure transmitted light through photodetector
Types of Photodetectors
Barrier-Layer Cell (Photocell)

● Also known as photovoltaic cell.
● composed of light-sensitive materials (e.g. selenium on
iron plate with transparent layer of silver).
● when exposed to light, electrons in the light sensitive
material are excited and are released into the highly
conductive silver.
● no need for power source.
● Electricity - stream of electrons.
○ Electricity is produced in Barrier layer cells is
once the light hits the light sensitive material,it
is excited. It jumps and is captured by highly
conductive silver.
● slowest response time.
● difficult to amplify electrical energy..
● inexpensive and durable,
● but temperature sensitive and nonlinear at very low or
very high levels of illumination ○ Cathode = photoemissive material
● thin metal film (transparent)
Photomultiplier (PM Tube)
● detects and amplifies radiant energy
○ incident light strikes the coated cathode,
emitting electrons
○ electrons are attracted to a series of anodes
(dynodes), each with successively higher
positive voltage
● dynodes give off many secondary electrons when hit by a
single electron
○ Dynodes - intensify the single light that is being
absorbed.
● Even at very low intensities of light, it will still give a
signal.
● used in instruments designed to be extremely sensitive to
very low light levels and light flashes of very short
duration
● rapid response time (10-15 dynodes); not subject to
fatigue
● Problem: Do not expose in room light as it will burn out.
Phototube
● A.k.a photoemissive tube
● also has photosensitive material that gives off electrons
when light energy strikes
● composed of a vacuumed glass that encloses:
○ positively-charged anode
○ negatively-charged cathode (e.g., Rb or Li)
● emitted electrons jump over to the positively charged
anode, where they are collected and return through an
external, measurable circuit
● Similar to barrier cell but requires an external source of
energy
11
○ Acts as a gap between p and n type
○ If there's a gap, the circuit is open→ no flow of
electricity.
● The amount of electrons is proportional to the amount of
photons hit
Photodiode (PDA)
● Arrangements of several photodiodes
● absorption of radiant energy by a reverse-biased
pn-junction diode (pn, positive-negative) produces a
photocurrent that is proportional to the incident radiant
power
● not as sensitive as PM, but with excellent linearity, speed
and small size
● A specialized diode which is sensitive to light. ● Specially used in post sample filter.
● It has excellent linearity ● If the monochromator is located before the cuvette =
○ Linearity - even at low light, the voltage also pre-sample filter
constantly increase in a linear fashion. ○ If after the cuvette = post sample filter
● Excellent response time ● U could arrange the arrays so that each of the colors are
● Small size being hit with only one wavelength.
● Spectral analysis.
Read-out device
● displays the amount of light transmitted
● examples:
○ Analog
■ needle along a scale
○ Digital
■ microprocessor to display results
using light-emitting diode (LED) of
liquid crystal display (LCD)
○ Recorder
■ strip chart or an integrator giving out
tracings
● Depletion Or intrinsic region

PART THREE
can now deduce if the spectrophotometer is
QUALITY ASSURANCE IN SPECTROPHOTOMETRY giving off tama na wavelength
● Wavelength or photometric accuracy ○ Accuracy = the closeness of a measurement to
● Absorbance check its true value
● Stray light
● Linearity Absorbance Check
● Making sure that the spectrophotometer is working ● Performed using glass filters or solutions that have
properly known absorbance values for a specific wavelength
● How to measure?
Wavelength or Photometric Accuracy ○ Use a specific glass filter again or a solution
● Implies that a photometer is measuring at the wavelength that has a known absorbance to check if the
that it is set to spectrophotometer corresponds to the
● Using special glass-type optical filters absorbance value as stated by the
(Examples:didymium and holmium oxide) manufacturer’s solution
● How do we know that spectrophotometer is really giving

off 340 nanometer?
○ We have to calibrate
○ We measure it against a standard
■ Didymium glass
● Which has a peak
absorbance at 600
nanometers
■ Holmium oxide
● Has multiple absorption
peak with a sharp peak
occurring at 360
nanometers
○ Using these special glass type optical filters, we
12
Linearity ● Remember: Percent transmittance is always less than
● Ability of a photometric system to yield a linear 100
relationship between the radiant power incident upon its
detector and the concentration (i.e., Beer’s law)
● There should be a 1 to 1 correspondence absorbance
and concentration
○ As the concentration of the analyte in the ● Transmitted light should always be less than or equal to
solution increases, the absorbance must also the incident light j
increase
● Linearity is ideal but we could not achieve linearity in Absorbance
practical sense ● Amount of light absorbed
○ If the concentration is too much, then the ● It cannot be measured directly by the spectrophotometer,
absorbance could not anymore increase but but rather is mathematically derived from the percent
rather it will plateau transmittance
■ Plateauing = non-linearity; does not ○ A = -log(%T)
anymore follow the line ○ A = log 100% - log (%T)
○ A = 2 - log (%T) → formula
■ This is already programmed in your
spectrophotometer
■ Spectrophotometer will determine the
percent transmittance and will derive
the absorbance
■ Mathematical relationship of the
absorbed light and the transmitted
light
● You can’t directly measure absorbed light because you
cant put an instrument inside the cuvette
● If the analysis is linear, the better
Beer’s Law
● But at some point, if the analyte is too much, it might
deviate from that linearity ● Absorbance is directly proportional to the concentration
● In some textbooks, it's actually A = abc
○ A - absorptivity
Stray Light
○ But, if the unit is in moles (molar), we use the
● Any light that impinges upon the detector that does not greek letter eta
originate from a polychromatic light source ● Absorbance is directly proportional to the concentration
○ Ex. if the spectrophotometer’s light is leaking (vice versa)
○ Make sure that the compartment that houses ○ The higher the concentration, the higher the
the spectrophotometer is protected from light absorbance and the lower the concentration,
bcs your photodetector might detect that stray the lower the absorbance
light and will create noise sa imong signal.make
deviations from the true value A = 𝜀𝑏c
● EMR that reaches the detector but is outside the narrow Where:
bandpass set by the monochromator ● 𝜀 - molar absorptivity
● Have a significant impact on any measurement made ● b - length of light path
○ Because we are measuring the concentration of ● c - concentration
the analyte by measuring the transmitted light
○ If we have additional light that reaches the ● Molar absorptivity - characteristic of analyte; ability of
photodetector, it might be miscomputed by your analyte to absorb light; measure it numerically
spectrophotometer as additional concentration ○ Molar absorptivity of a given analyte is constant
or false decrease in the analyte ○ Ex. Glucose of DNS
■ glucose has its own molar absorptivity
PRINCIPLES OF SPECTROPHOTOMETRY which is constant regardless of the
concentration of glucose -- even if
Beer’s Law (Beer-Lambert Law) con. is high or low, MA is constant
● The concentration of a substance is directly proportional ● Length of light path - the path of light; path that light
to the amount of light absorbed and inversely travels inside the cuvette; coming from the incident light
proportional to the logarithm of the light transmitted and when the transmitted light first goes out of the wall of
● Law that guides the principle of spectrophotometry the cuvette
● 3 lights of concern: ○ In any given analysis of spectrophotometer, the
○ Incident light - coming from the light source to length of light path is also constant because it
the cuvette doesn't make sense that cuvette will change in
○ Absorbed light shape or length (1cm forever)
○ Transmitted light ● Concentration - not constant
● Directly proportional = the higher the concentration, the ● Absorbance is proportional to the concentration;
higher the absorbed light absorbance is dependent
● Inversely proportional = whenever the concentration is
high, the transmitted light is low and vice versa. Standard
● A solution containing a precisely known concentration of
Percent Transmittance an element or a substance
● The ratio of the radiant light transmitted divided by the
𝐴𝑢 𝐶𝑢
●
radiant energy incident to the sample
Tells u how much in percent of the incident light passed 𝐴𝑠𝑡𝑑
= 𝐶𝑠𝑡𝑑
through the cuvette
𝑡𝑟𝑎𝑛𝑠𝑚𝑖𝑡𝑡𝑒𝑑 𝑙𝑖𝑔ℎ𝑡
%𝑇 = 𝐼𝑛𝑐𝑖𝑑𝑒𝑛𝑡 𝑙𝑖𝑔ℎ𝑡
𝑥 100 ● Standard solution - solution that has known concentration
○ Ex. Glucose std = 100mg/dl
13
○ Sample (unknown) put in the ● Spits or chops the monochromatic beam of radiation
spectrophotometer, you’ll get the A u into two components:
○ Standard, put same amount reagent, and then ○ One beam passes through the sample, and the
spectrophotometer, you’ll get the A std other passes through a reference solution or
○ 100 mg/dl = Cstd blank
○ Compute for the Cu ● Two designs:
○ Double beam in space
○ Double beam in time
Ex.
● Sample (Au) = 0.5 Review:
● Std (Astd) = 0.25 ● What really happens inside the Clin Chem Lab when you
● Cstd = 100mg/dl do spectrophotometric analysis
○ Extract patient sample
𝐴𝑢 𝐶𝑢
𝐴𝑠𝑡𝑑
= 𝐶𝑠𝑡𝑑
○ Process blood sample to get serum/plasma
○ 1st cuvette: Serum + reagent → rxn
0.5 𝑥 ○ 2nd cuvette: Standard + reagent → rxn
0.25
= 100𝑚𝑔/𝑑𝑙 ○ 3rd cuvette: blank
0.5 (100mg/dl) = 0.25x ● To do it manually
200mg/dl = x ○ First, measure the absorbance of the blank
○ Assuming your blank is 0.02, depending on the
The amount of glucose in the plasma of the patient is spectrophotometer
200mg/dl. ○ There are certain spectrophotometers that after
you read the absorbance of the blank, all you
have to do is press “return to zero”
○ Everything that is being read is automatically
Blank
deducted by 0.02
● Solution containing no analyte of interest, usually used to ○ After deducting and returning to zero, you
calibrate instruments read the absorbance standard
○ Reagent blank ○ Remove the cuvette that houses the standard
○ Water blank and then you put the next cuvette with the
○ Air blank serum + reagent = 0.25
● Let's say you have a weighing scale, how will you weigh ○ Do the computation of
a baby?
𝐴𝑢 𝐶𝑢
○ You can step on the weighing scale while 𝐴= 𝐴 𝑠𝑡𝑑
= 𝐶 𝑠𝑡𝑑
carrying the baby -- 100kg
○ And weigh yourself without the baby -- 94kg ○ In a single beam spectrophotometer,becuse
○ 100kg - 94kg = 6kg weight of the baby there is only one slot ofr the cuvette, you need
○ How you measure the baby, without directly to place it one by one
measuring the baby
● Ex. 2 You want to measure fish in the timbangan
○ You can directly place the fish in the timbangan
but you have to have a holder
○ Measure the weight of the holder first, then
measure it with the fish
○ Subtract the weight of the holder with the total
weight of both the holder and the fish to know
the weight of the fish
● Ex. 3 Water blank - just water and nothing else
○ In your analysis, you have to add d. Water
■ The water itself absorbs a particular
wavelength
■ Sample + reagent (reagent contains
water)
○ In a given incident light absorbed, and the Double Beam in Space
absorbance is 0.90
○ When you use water blank, with the same
wavelength
■ Absorbance of just water is 0.01
○ To know the absorbance of the sample and the
reagent, without the water, you have to subtract
○ 0.90 - 0.01 = 0.89 true absorbance of the
sample and the reagent (w/o water)
● Ex. 4 Reagent blank - use reagent with no sample
○ Reagent - test kit; a chemical you add to your
sample to generate a reaction to measure the
analyte
○ After measuring, the absorbance is 0.56
○ Then, you do a regent blank and measured the
reagent as 0.06
○ The true absorbance of the sample is not 0.56
but minus ang blank 0.50
● In reality, once you loaded the blank in your ● Has two photodetectors
spectrophotometer, it will automatically subtract the ● There is a light source but there is a beam splitter
absorbance of the blank ○ Splits the beam into the sample and the other
○ Blank will depend on the analyte goes to the standard cuvette
DOUBLE-BEAM SPECTROPHOTOMETRY ○ Using two light sources is prove to error since
14
you cannot ensure that both sources will emit
the same intensity of light
○ After reading the absorbance, the
spectrophotometer will automatically compute
for the concentration
● used for Group 1 metals (1+ charge)

● Na+ , K+ , Li+
○ ‘excitable’ metals that emit a specific light
● Na+ – emits yellow light (589 nm)

Double Beam in Time ● K+ – emits only violet light (367 nm)
● Li+ – transmits only red light (767nm)
Components of FES
● Flame
○ breaks the chemical bonds to produce atoms
○ source of energy that will be absorbed by the
atoms to enter the excitation state
○ also serves as the cuvette
■ No cuvette
● Atomizer
○ breaks up the solution into finer droplets so that
the atom will absorb heat energy from the flame
and get excited
● There is only one photodetector ○ Does not produce atoms
● There are still double beam ○ Smaller droplets are easier to burn
● But because there is a single photodetector, we have to ● Burner
use a rotating chopper ○ types:
● It is a mirror that the spectrophotometer rotates so that if ■ total consumption burners
you want to read the sample cuvette’s absorbance, yan ● the sample is aspirated
ang i bounce sa photo detector and once it roatttes, it ill directly into the flame
block the light form the sample cuvette and read the light ● the flame can be made
from the standard cuvette hotter but there is the
production of large droplets
FLAME EMISSION SPECTROPHOTOMETRY in the flame
● Or Flame Emission Photometry ■ premix burner
● Sometimes FES or FEP ● the sample is atomized
● The measurement of emitted light when electrons in an before entering the flame
atom become excited by heat energy produced by the and does not create noise
flame ● gravitational feeding of
● Excited atoms return to the ground state by emitting light sample into the flame
energy that is a characteristic of that atom ● Interference Filter
● When light strikes an electron, when a given wavelength ○ Filter is found after the flame
of light is being absorbed by the electron, the electron ○ Na+ filter – transmits only yellow light (589 nm)
becomes excited ○ K + filter – transmits only violet light (367 nm)
● After the excitement, it relaxes ○ Li + – transmits only red light (767nm)
● Once the electron relaxes back to its original level, it
reemits the light it has taken up previously
● That particular emission of light is what we measure for
us to know the concentration of the given element
● Main parts of FES:
○ Sample injection Port
○ Acetylene Flame Source ● The more violet light, the higher the potassium is
ATOMIC ABSORPTION SPECTOPHOTOMETRY

● measures concentration of element by detecting
absorption of light (EMR) by atoms
● elements are not excited but they are dissociated from
their chemical bonds and placed in the unionized,
unexcited ground state
● AAS is used for group 2 metals
○ Calcium and Magnesium
15
● used for Group two metals (2+ charge)
● not easily excited but can absorb light
○ Higher temperatures are required for them to
be excited
● Just like Sodium, Potassium, and Lithium wherein they
can absorb light so the electrons are excited but once
they are in a relaxation state, they re-emit the same
amount of light they absorbed
● We have a:
○ Burner
○ light source (hollow cathode)
○ chopper which turns a single beam into a
pulsating beam (it closes the beam to be turned
on and off)
○ Monochromator which will select a particular
wavelength of light after the burner (just like
how we put the interference filter in FES)
○ PM tube is protected by the monochromator
● We have two sources of light going to the PM tube
○ 1st light: light coming from the excited atoms
■ But, it is unexcited if group 2 metals
● Serum aside from
containing magnesium and
calcium, it also contains
potassium and sodium
● Components ● so it is inevitable to have
○ Burner excitation in this flame (acts
■ uses a flame to dissociate the as an FES which emits light
chemical bonds and form free coming from the excited
unexcited atoms atoms)
■ serves as the cuvette ● It is a problem since we
○ Monochromator don't want that emitted light
■ selects the desired wavelength from a coming from excited atoms,
spectrum of wavelength but the absorbed light from
■ Because it is located after the burner, those unexcited atoms in
it is also a post-sample filter Group 2
■ Function: serves to protect the ○ 2nd light: Hollow cathode lamp
photodetector from excessive light ■ Light source
coming from the flame ● If the chopper closes and cuts off the light from the light
■ Why? source
● PM tube burns out ○ So, the PM tube detects (A) light emitted from
whenever exposed to the excited atoms
excessive light ● If the chopper is open, the PM now detects both lights
○ Photodetector emitted from the excited atoms and hollow cathode lamp
■ Photomultiplier (PM) tube ○ The amount of light being absorbed is
○ read-out device indirectly proportional to the transmitted light
■ The amount of light that is being ● What the computer does is to subtract the detection
absorbed by the Group 2 metals amount of light when the chopper is open (1+2) with
● Ex. The higher the when the chipper is closed (1)
magnesium, the more light ○ The readout device will only give you the
is absorbed amount of light that is emitted by the light
■ The transmitted light that reaches source (=2)
your photodetector is low ● We are basically eliminating the light coming from the
Group 1 metals (excited atoms)
● Interferences
○ Chemical
■ situation at which the flame could not
dissociate the sample into neutral
atoms.
■ Ex. calcium
● Calcium phosphate
○ U put something
into the serum first
so that it could
dissociate into
calcium molecules.
○ ionization
16
■ situation at which atoms in the flame
become excited and emits energy.
○ FES is obsolete as well as AAS (ref method).
Types of Photometric Instruments
● Spectroscope
● Colorimeter
● Photometer
● Spectrometer
Spectroscope
● an optical instrument used for visual identification of
atomic emission lines
● has a monochromator, (prism or diffraction grating)
● exit slit is replaced by an eyepiece that can be moved
along the focal plane.
● Direct it in your eye and detect the spectral line.
Colorimeter
● Matamater
● Subjective
● uses the human eye as the detector
● user compares the observed color of the unknown
sample against a standard or a series of colored
standards of known concentrations
● Ex: urine dipstick
Photometer
● consist of a light source, a filter, and photoelectric
transducer, as well as a signal processor and Readout.
○ Photoelectric transducer = photodetector
● some manufacturers use the term colorimeter or
photoelectric colorimeter
● use filters for isolation of specific wavelengths, NOT
gratings or prisms
Spectrometer
● an instrument that provides information about the
intensity of radiation as a function of wavelength or
frequency
● spectrophotometer are spectrometers equipped with one
or more exit slits and photoelectron transducer.
● Detects whole spectrum of light.
● No monochromator.
17
LECTURE 5: LUMINESCENSE
Prof. JC LOuise Bandala, RMT
September 6, 2021
LUMINESCENCE both electrons get combined, like a magnet = triplet state

● is the emission of light by a substance ● In molecule, there are several electronic levels (electrons
● occurs when an electron returns to the electronic ground are present at the ground state) in the singlet state
state from an excited state and loses its excess energy (opposite spin)
as a photon ● Fluorescence phenomenon
● Three types: ○ When molecules absorb UV or visible light, one
○ Fluorescence electron from ground state will go to an excited
○ Phosphorescence state
○ Chemiluminescence ○ Excited state is having several vibration levels ,
There are substances that when u give heat, it will absorb the heat excited electron loses energy by intermolecular
and gets excited away from its normal orbit and jump off another collision and comes to the lowest vibration level
orbit. Pero dili man niya forever i carry ang certain heat/energy na ○ It then returns to ground state by emitting the
nakuha niya. Thus, there will come a time na mubalik siya didto sa radiation or light of lower energy or higher
iyang orbit. Pagbalik niya sa original na orbit, kailangan niya i give wavelength
off ang energy. Upon giving off that energy, mao to ang light ○ Instant re-emission of light
emitted. ○ Energy of radiation emitted is always less than
the energy of radiation absorbed
FLOURESCENCE ○ Wavelength of emitted radiation will always be
● When a beam of light is incident on certain substances, higher than the wavelength of radiation
they emit visible light or radiations absorbed
● It starts immediately after the absorption of light and ● Phosphorescence
stops as soon as the incident light is cut off ○ Inversion of electron spin takes place
○ When the energy from UV or visible light is
absorbed, the electron from the ground state
jumps to excited state, at the same time the
spin of electron gets reversed and it is now in
triplet state (same spin)
○ Pairing of electron with same spin is not
favorable thus it takes more time to reverse the
spin and come back to ground state
○ Delay re-emission of radiation
○ The energy of radiation emitted is always less
● Upon directing that light into your substance, it will than the energy of radiation absorbed
immediately absorb it. ○ Wavelength of emitted radiation is higher than
● By the time na tanggalon ang light source, mawala lang the wavelength of radiation absorbed
pud ang incident light
PHOSPHORESCENCE
● Also known as Delayed fluorescence
● When light radiation is incident on certain substances,
they emit light continuously even after the incident light is
cut off, it will remain.
● By the time na naay light na irender padulong sa

substance, iabsorb to niya na energy. ● Fluorescence is immediate emission of light;
● By the time we remove the source, it will continue to glow phosphorescence is delayed
or absorb the light (longer period of time) ● Process that involves transition of electronic state (singlet
○ But if you give it some time, it will go back to its and triplet state) is called intersystem crossing
original appearance ● Having different state of spin is the reason why
○ fluorescence - immediate phosphorescence takes time (delay)
○ Phosphorescence - minutes or days
● Difference between phosphorescence and fluorescence HOW TO MEASURE FLUORESCENCE?
○ Instant re-emission of the absorbed energy is ● Fluorometry
called fluorescence ○ Measures the fluorescence or the energy
○ Delayed remission of the absorbed energy is emission that occurs when a certain compound
called phosphorescence absorb electromagnetic radiation, become
excited and then return to an energy state that
● pair of electrons have opposite spin = singlet state is usually higher than their original level
● Electrons having the same spin, magnetic moment of ○ The emitted fluorescent light has longer
1
wavelength and lower energy which is due to for both absorption and fluorescence
the energy lost between the time when the ○ Fluorescence measures the amount of light
photon is absorbed and when it is emitted intensity present over a zero background
○ Emitted light has lower wavelength and lower ○ Zero background- lesser interference occurs
energy - due to energy loss that happened Disadvantages
between the time it absorbed photon and ● Very sensitive to environmental changes
energy is emitted ● Quenching- quick disappearance of fluorescence
○ Changes in pH affect electron availability
Basic Components of Fluorometry ○ Temperature changes the probability of loss of
energy
● Light source ○ Contaminating chemicals or a change of
○ Mercury vapor lamp (commonly used) or solvent may change the structure
xenon arc lamp ○ UV light used for excitation can cause
● Excitation/Primary Monochromator photochemical changes especially if dili gina
○ Selects the wavelength that is best absorbed calibrate every now and then
by the solution to be measures ○ Fluorometry is not often used nowadays
○ Monochromator: one single color; from a wide
range of colors, it narrows down an individual CHEMILUMINESCENCE
color or wavelength selected ● Is the production of light from a chemical reaction
● Cuvette ○ From the word “chemi”- there is a chemical
● Emission/Secondary Monochromator reaction that happened inside
○ Filters out fluorescence form stray light ● Reactions are oxidation reactions of luminol, acridinium
radiation steers, and dioxetanes characterized by a rapid increase
○ Position at a right angle from the cuvette to in intensity of emitted light followed by a gradual decay
eliminate potential interference from the ○ Other examples na most common: oxygen,
excitation light hypochlorite, hydrogen peroxide
○ What we are trying to capture is the emitted ● The excitation of the substance does not involve
light already electromagnetic radiation and no monochromators are
● Photodetector needed, instead, the excitation energy comes from a
○ The commonly used photodetector is the PMT chemical or electrochemical reaction
or Photo Multiplier Tube ○ The most basic difference between
○ Because it has the highest sensitivity compared fluorescence, phosphorescence, and
to other photodetectors chemiluminescence
○ In chemiluminescence, the presence of
radiation is not necessary, in measuring light
absorbed dili kailangan ug monochromator
purely chemical reaction lang
● The light signal is measured against a completely dark
background
○ Nganong kailangan i break ang glowstick?
■ Naa duha ka compartment
■ Naay inside and outside na portion
■ Each compartment has different
chemicals inside
■ That is why it is necessary to break
the glowstick para naay interaction
between those chemicals presence
creating a glowing light
(VIDEO)
● There are reactions where light is emitted without the
emission of a considerable amount of heat. This
● The secondary filter is positioned at a 90-degree angle phenomenon is called cold light or chemiluminescence
● Attenuator - another term for entrance slit ● In the following experiments chemiluminescence
● Why is positioned at 90 degrees? accompanying the oxidation of luminol is demonstrated.
○ Remember your transmitted light, and diba dili ● A solution of ammonia water NH3+h20 with potassium
transmitted light ang atong gina try measure diri tricyanocuprate(I) K2 (Cu(CN)3) addition has been
buy tour emitted light prepared
○ The emitted light is not directly released from ● To oxidize the luminol solution of hydrogen peroxide,
your sample holder H2O2 is added to the mixture
○ Examples of substances that can absorb light ● A more generous amount of H2O2 added
and can cause fluorescence ● Luminol was oxidized by hydrogen peroxide, energy was
■ POPOP- phenyloxizolebenzene emitted in the reaction as light quanta
■ Quinine ● Several consumer goods based on luminescence are
■ Fluorescein available on the market
■ Acridine Orange ● Different light colors are obtained with different dyes
■ Rhodamine B ○ Blue- 9,10-Diphenylentracene
■ Pyridine 1 ○ Orange- 5,12-Bis(phenyl ethynyl)naphthacene
○ The term for the atom molecule that can cause ○ Red- Rhodamine B
fluorescence is a fluorophore ○ Yellow-green-1-Chloro-9,10-bis(phenyl
ethynyl)anthracene
Advantages ● Chemiluminescence can be observed in nature. For
● Increased sensitivity (1000x more sensitive than example, the green glow of the common glow-worm is
spectrophotometric methods) known
○ Emitted radiation is measured directly ● Luciferin is the dye here, activated by enzyme luciferase
● Increased specificity by selecting the optimal wavelength and air oxygen
● ADVANTAGES:
2
○ Subpicomolar detection limits
○ Speed (Fast)
■ Flash-type reactions
■ Light s measured for 10 seconds
○ Ease of use (mix of chemicals only)
○ Simple instrumentation
● DISADVANTAGES:
○ Impurities can cause a background signal that
degrades sensitivity and specificity
NEPHELOMETRY AND TURBIDIMETRY

● Light scattering is a physical phenomenon that results
from the interaction of light with particles in solution.
● Unlike fluorescence emission, the wavelength of the
scattered light is the same as that of the incident light.
NEPHELOMETRY Nephelometry Turbidimetry

● measures the amount of light scattered in a particulate Mercury arc lamp Tungsten lamp
suspension at 90-degree angle Scattered light is measured Light transmitted is measured
● useful method to determine the concentration of solutions
Measured at 90 degrees Measured in straight line
that contains particles too large for absorption
PMT is the detector Photocell is the detector
spectrometry
○ Applicable to both 4
● Too large particles but very low concentration FACTORS THAT AFFECT SCATTERED LIGHT
○ If light is introduced, it tends to scatter it out ● Particle size
● The detecting cell is placed at right angles to the light ○ The smaller the particles, the scattered the light
source, to measure light scattered by particles. The ● Concentration of particles
intensity of the scattered light savers as a measure ● Molecular weight of particles
of the turbidity. The instrument is called a ● Wavelength dependence
“Nephelometer” or a “Nephelometric Turbidimeter”. A
spectrophotometer can be used, however, a special THREE TYPES OF SCATTERED LIGHT
attachment is required for nephelometry.
Rayleigh
TURBIDIMETRY
● Turbid - cloudiness or haziness of the solution
● measures the amount of light blocked in a particulate
suspension
○ ↓ in light transmission
● Amount of light blocked depends not only on
concentration but also on the size
● Sampling handling becomes critical
○ Since the particles in the sample are too large
and tend to aggregate with each other, so dali
mag settle down ang suspension ● Wavelength of light > particle size
○ So before measuring the substances, we really ● Light symmetrically scattered around the particle.
need to mix samples well first to prevent ● Answers as to why the sky is blue.
miscalculation ● Amount or intensity of the scattered light is inversely
● Thou large particles, its scattering is extensive = hazy proportional to the 4th power of the wavelength of a
sample - high concentration wave.
○ Once light is present, it will not be able to pass ● If the wavelength increases, intensity will be lower.
through because of the haziness of the ● Lesser wavelength, more scattering.
suspension
● The amount of light passing through a solution is Mie
measured. The higher the turbidity, the smaller the
quantity of light transmitted (i.e. more light i absorbed).
Any spectrophotometer or photometer can be used as a
turbidimeter, without modification. Since property
concerns visible light the measurement is commonly
carried out at 420 nm.
● Wavelength of light < particle size

● Light scatters backward but appears forward due to
destruction out of phase background scatter
● Explains why the clouds are white
● Light scattering in all directions.
Rayleigh Debye
3
● Wavelength of light = particle size
● More forward light scatter
● Antigen-antibody reactions
● Mas daghan ang scattered light sa rayleigh debye
compared to Mie
4
LECTURE 6: CHROMATOGRAPHY AND MASS

SPECTROMETRY
Prof. JC LOuise Bandala, RMT
September 6, 2021
CHROMATOGRAPHY GAS CHROMATOGRAPHY

● is an analytical technique commonly used for separating ● separating compounds based primarily on their volatility
a mixture of chemical substances into its individual ○ Volatility = easily evaporated at normal
components, so that the individual components can be temperature
thoroughly analyzed ● is useful for compounds that are naturally volatile or can
○ All about separation be easily converted into a volatile form
○ Chrome = color ○ Thus, dili tanan compounds pwede gamiton ug
gas chromatography
BASIC COMPONENTS OF CHROMATOGRAPHY ● Two types:
● Mobile phase or carrier ○ Gas-Liquid Chromatography: based on partition
○ Gas or liquid ○ Gas-Solid Chromatography: based on
○ Solvent moving through the column adsorption
○ Carries the sample
● Stationary phase or absorbent
○ Solid or liquid
○ Where the mobile phase flows
○ Does not move; fixed
● Column – holds the stationary phase
● Eluate – separated components
● Eluent – Fluid or substance that enters the column and
moves the analyte; help in the movement of analyte
● Elution – The process of washing out a compound
through a column using a suitable solvent
● Analyte – Mixture whose individual components have to
be separated and analyzed
● Retention time or factor – The time it takes for a
compound or analyte to elute
● Butanganan = column
● Stationary phase = Materials inside (beads) ● Based on the vid, your sample is being evaporated
● Mobile phase = what will pass to ur column carrying ur ● May collector sa may detector
analyte ● FID = Flame ionization detector
● Analyte = shaded violet sa top ○ able to detect whatever sample was vaporized .
● Once u place your analyte, and then naa moy makita na ● Any changes na mahitabo sa FID is being recorded
white na part (eluent) na in order for your sample to ● Peak = referring to the concentration of your sample
move and start being separated
● After some time from your original analyte, your BASIC COMPONENTS OF GAS CHROMATOGRAPHY
components will now separate
● Columns
● In terms of who comes out first and who comes out last,
○ Packed columns or Capillary columns
it would depend on what are the substance that u used
○ Glass or stainless steel (packed) or thin-fused
● Chromatographic techniques may be classified according
silica (capillary)
to their mobile phase:
○ Packed columns are filled with inert particles
○ Gas chromatography
such as diatomaceous earth or porous polymer
○ Liquid chromatography
or glass beads coated with a nonvolatile liquid
(stationary) phase
1
○ liquid stationary phase must be nonvolatile at gas and the sample gas which produces an
the temperatures used, must be thermally electrical signal unique to the compounds being
stable, and must not react chemically with the analyzed
solutes to be separated ○ This signal is proportional to the concentration
■ May cause wrong detection of certain of the sample components provided a direct
substances means of measuring component concentrations
○ Packed column in a particular sample and form this you receive
■ Packed inside a chromatogram that represents the
■ Packed up in the hollow portion (could components found in the sample analyzed
be bead column or porous column ● Additional input on how TCD work:
layer) ○ Wheatstone bridge - measures unknown
■ Used for gas-liquid chromatography resistance values in the TCD
or gas-solid chromatography ■ Can also be used for calibration of
■ Greater sample capacity different instruments
○ Capillary column ■ Important in the TCD
■ Open column ○ Carrier gas used is helium
■ Hollow part in the column is empty ○ 2 entries
■ There's coating of the sides of column ■ One where the sample enters and the
■ Used only for gas liquid other for standard reference
chromatography ■ As your reference and sample enters
■ Can provide higher separation each compartment, once we
efficiency compared to packed introduce heat, sample components
column have lower thermal conductivity
■ Can be overloaded easily compared to reference
■ Because of difference in thermal
TWO DETECTORS conductivity, it will create changes in
● Thermal conductivity the electrical resistance detected by
○ Contains wires (filaments) that change the filament
electrical resistance with change in temperature ■ That electrical change is directly
● Flame ionization detector proportional to the concentration of
○ More sensitive that TC detectors your analytes causing peaks in your
○ Small hydrogen flame and collector electrode chromatogram
■ Collects specific particle or molecule
○ As the sample burns, ions form and move to
the charged collector
● Flame Ionization Detector

○ Important component: have a hydrogen supply
○ Sample inlet - where your sample enters
○ Collector electrode
○ Flame ignitor
○ Almost universally employed, where the flame
commonly is generated with hydrogen and air
○ The autosampler provides the means to
introduce a sample automatically into the inlets
■ Manual insertion is also possible
○ The column inlet or injector is attached to the
column head and provides the means to
introduce a sample into a continuous flow of
carrier gas
○ In the injector, a sample is introduced to a
● How does a TCD work? heated chamber via a syringe through septum
○ Each gas enters the gas the gas ○ Heat facilitates volatilization of the sample and
chromatograph instrument separately sample matrix
○ The sample goes into one column while the ○ Carrier gas then either sweeps the entirety
pure carrier gas goes into another column splitless mode or a portion split mode of the
○ Electrically heated resistance wires are located sample into the column
in chambers inside of the TCD ■ Split mode - a part of the sample
○ Power supply provides a current to the carrier gas mixture in the injection
resistance wires which causes the wires to heat chamber is exhausted through the
up split vent
○ The electrical circuitry shown here is a ■ Splitless mode - all the sample carrier
characteristic of thermal conductivity detectors gas mixture in the injection chamber
(TCD) which is known as the wheatstone bridge is transported through the column
○ As the gas flows through the TCD, the physical ○ 2 types of column:used in GC
properties of the reference and sample gases ■ Packed column - where the stationary
(ex. Specific heat capacities) will allow the phase is coded directly in the column
wired of the TCD to be cooled at different rates, ■ Capillary column - where the
this change in temperature will result in a stationary phase is coded with the
change in resistance from both the reference inner wall of the column
2
○ Mixture separation is based differential Types of Separation Chromatography
partitioning of components between the mobile ● Adsorption
and stationary phases ● Partition
○ The component with less affinity to the ● Steric exclusion
stationary phase, consequently less interaction ● Affinity
travels faster and diluted out first ● Ion-exchange
○ The component which has more affinity to the
stationary phase, consequently more Adsorption
interaction travels slower and diluted later
● Liquid-solid chromatography
○ In addition, other factor influence the separation
○ Adsorption- adhesion of atoms or your
of the components such as column
molecules to a certain surface
temperature, carrier gas flow rate, column
○ First word= mobile phase
length, amount of material injected
○ Second word= stationary phase
○ As compounds elute from the column, they
● Competition between the sample and the mobile phase
interact with the detector
for the adsorptive sites on the solid stationary phase
○ Different detectors can be used:
● Stationary phase can be acidic polar (e.g., silica gel),
■ Flame ionization detector
basic polar (e.g., alumina), or nonpolar (e.g., charcoal)
- Based on the detection of
○ Depends on the analyte of interest kug asa siya
ions formed during
better mag adhere
combustion of organic
● Disadvantage: strong retention of many compounds by
compounds in a flame which
the supports, making them difficult to elute from the
generated by hydrogen and
column
air
- To detect these ions, 2
electrodes are used to
provide a potential
difference
- The positive electrode
doubles as a nozzle head
where the flame is produced
- The negative electrode is
positioned above the flame
- When another compound is
mixed with the hydrogen
flame, mainly carbon ions
are generated (VIDEO)
- Current is produced ● Based n the fact that certain solid materials known as the
between the electrodes adsorbents have the ability to hold molecules on the
proportionally to the amount surface by the phenomenon of adsorption
of organic compound ● The adsorption process usually involves attracted forces
present like Van der wals interaaction and hydrogen bonding
- This current is measured ● One of the common adsorbent ifs silica
with an electrometer ● Silica has silanol, Si-OH functional group
amplified into proper voltage ● The Si-OH group interacts with other functional groups
and fed into an integrator of the sample molecules
- Number of peaks indicate ● Different sample molecules bind the absorbent with
how many components are different affiinitty
in the mixture ● Hence when the mobile phase passes the molecules
■ Thermal conductivity detector having less interaction with adsorbents are release first
■ Mass spectrometer detector while the molecules having the most interaction will be
○ X-axis of gas chromatogram shows the amount released last
of time taken for the analytes to pass through ● Adsorption, adherence, affinity to stationary phase
the column and reach the FID detector
○ Y-axis (area of the peak) - a reflection of the Partition
amount of a specific analyte present ● Liquid-liquid chromatography
● Separation of substances according to their solubility in
LIQUID CHROMATOGRAPHY an organic/non-organic polar solvent and in an
● Uses lower temperatures for separation achieving better aqueous/polar solvent
separation of thermolabile compounds ● “Like dissolves like”
○ Thermolabile- compound is unstable when ● Polar molecules remain in the aqueous solvent; nonpolar
heated and they are readily destroyed or molecules are extracted in the organic solvent
deactivated when introduced to heat
● Easier to recover a sample compared to GC
● Commonly used and suitable alternative than GC
○ Because not all compounds are not volatile.
Some are too unstable, insufficiently volatile to
be assayed using your gas chromatography
● The mobile phase can be removed, and the sample can
be processed further or reanalyzed under different
conditions
● Kaya siya tinatawag na paper chromatography because

the support used is a paper
3
● Instead of using a marker, what is used is a leaf extract in Resin Type Cation Exchanger Anion Exchanger
a paper Net charge of a
● Paper is the support and the leaf extract is the liquid molecule of interest + -
● The other liquid is the propanone (acetone) which will
serve as your mobile phase that will carry your analyte Change of resin - +
and then such time we see a reaction
● Solvent front refers to the distance moved by your Affinity
solvent from its starting point to the end ● most selective type of chromatography employed
● Observe for the solubility ● utilizes the specific interaction between one kind of solute
● The more soluble the component, the faster the molecule and a second molecule that is immobilized on a
movement, the greater the distance travelled stationary phase
● Since lahi-lahi man siya ug solubility, thuss, a separation ● Examples: antigen and antibody, enzyme and substrate,
will occur receptor and ligand, protein and nucleic acid
● Ethanol, methanol, or chloroform canalso be used as ○ Similar to lock and key
your mobile phase ○ Have specificity
Steric Exclusion
● Variation of L-S chromatography
● A.k.a. Size exclusion chromatography
● Separation based on size and shape
● Solid-phase is packed with porous material (beads) that
separates solutes according to size
● Similar to enzymes (specific)

(VIDEO)
(VIDEO) ● Affinity Chromatography occurs based on the specific
● No adsorption interaction between the two molecules
● No interaction between the molecules and the surface of ● This specific interaction can be between enzyme and the
the porous particles substrate, receptor and ligand, protein and nucleic acid,
● Size exclusion chromatography separates molecules etc
based on their size Components of Affinity Chromatography
● Smaller particles have a longer residence time in the ● Carried out in column which is filled with the supporting
column and larger molecules have a shorter residence material called matrix
time ● Matrix
● Longer column ○ Chemically inert
● SEC can be used for protein desoldering ○ Good flow
○ Salt ions are small and can enter into the pores ○ Have functional groups for the covalent binding
and gets longer residence time of the ligand
○ Proteins will have a shorter residence time ○ Agarose, polyacrylamide, polystyrene,
Residence time = retention factor cellulose, silica
● SEC vs Adsorption chromatography ● Matrix is attached with a space m which is usually made
○ SEC has no affinity up of Ch2 group
● The presence of space prevent the non-specific
Ion Exchange interaction of the ligand with the matrix itself
● use of a resin (stationary solid phase) for covalent ● Molecules that passed through the column and did not
attachment of anions or cations onto it bind will be washed away
● Solute ions of the opposite charge in the mobile liquid Removal of a molecule of interest from the column
phase are attracted to the resin by electrostatic forces ● Buffer with different pH or ionic strength is passed
● Widely used for the separation of proteins, peptides, and ○ This releases the molecule of interest and will
nucleic acids be obtained in a pure form
○ Proteins have charge ● Competitive inhibitor
○ Can be removed by dialysis or by changing the
pH od ionic strength
4
● As a result, efficiency of separation increases giving high
CHROMATOGRAPHIC PROCEDURES resolution
● Thin-layer chromatography ● Components;
● High-performance liquid chromatography
○ most recommended because it has highest
sensitivity
THIN-LAYER CHROMATOGRAPHY
● Variant of column chromatography
● A thin layer of sorbent, such as alumina, silica gel,
cellulose or cross-linked dextran, is uniformly coated on a
glass or plastic plate
● Most commonly used as a semiquantitative screening
test
○ column: made up of stainless steel which can
withstand a very high pressure upto 50 MP;
length can vary from 5 - 25 cm and have an
internal diameter of 4.5 mm
■ The flow rate from the mobile phase
to the column is usually 1-3 mL/min
● Almost the same with paper chromatography

● Mobile phase: where we put the analyte and after some
time through capillary action, there is separation
happening ○ Stationary phase: made up of an absorbent
9 material has a very small particle size and kept
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY uniform to obtain a better performance
● Uses pressure for fast separations, controlled ■ Usually chemical modified silica,
temperature, in-line detectors and gradient elution divinyl benzene and etc. are used as
techniques a SP
○ Mobile phase
■ Mixture of different solvents (polar or

nonpolar) which depends on the type
of molecule
■ Usually kept solvent reservoir
● attached to the pump which
pumps the mobile phase in
the column with high
pressure
■ Injector
● Just before the HPLC
column which introduction of
the sample into the column
■ Detector
● In order to detect the
● Modified column chromatography molecules of the sample
● A column is with speck with an absorbing material like that is coming out from the
silica and the mobile phase is pressed down the column column
because of gravity ● Types: UV, iode,
● But in HPLC, a high pressure pump is attached with a fluorescence, reflective
column which can generate a pressure up to 40 MP index detectors and
● The column is filled with an absorbing material which has spectrometers
a very small particle size and this gives a large surface ● Working: as the sample molecules get separated, they
area for the molecules to interact are detected and the peak is obtained from on the
5
computer which is plotted with respect to the retention
time
● To identity the components, we need to have standards
(serves as a basis)
○ Ex: if we run glucose as our sample and its
peak is obtained at 5 minutes. Next we run
sucrose, and its peak is gained at 8 minutes.
Now we analyze the unknown sample for the
detection of sugars present on it.
● Detectors
○ Monitor the eluate as it leaves the column
○ Produce an electronic signal proportional to the
concentration of each separated component
○ Spectrophotometers – detect absorbances of
visible or UV light
○ Photodiode array – used for spectral
comparisons and compound identification and
purity and for drug analysis in urine
○ Amperometric or electrochemical detector –
measures current produced when the analyte of
interest is oxidized or reduced at some fixed
potential set between a pair of electrodes
● Recorders
○ Used to record detector signal versus the time
○ This indicates that there is glucose but no mobile phase passed through the instrument,
sucrose. starting from the time of sample injection
○ The graph is called chromatogram
BASIC COMPONENTS OF HPLC ■ Peak area is proportional to
● Pump concentration of the compounds that
○ Forces the mobile phase through the column at produced the peaks
a much greater velocity
○ Helps in hastening separation MASS SPECTROMETRY
○ Types: ● always coupled to liquid or gas chromatography.
■ Mechanical reciprocating pump – ● Considered as the gold testing
most widely used ● It doubles the sensitivity and specificity = the better.
■ Pneumatic pumps – for preoperative ● based on fragmentation and ionization of molecules
purposes using a suitable source of energy
■ Hydraulic amplifier pumps – no longer ● charged particles moving through a magnetic or an
commonly used electrical field can be separated from other charged
● Columns particles according to their mass-to-charge (m/z) ratios.
○ Long stainless steel
○ Silica gel – most common BASIC COMPONENTS OF MS
● Sample injectors
○ Can be used to introduce the sample into the
path of the mobile phase that carries it into the
column
○ Loop injector
■ Best and most widely used
■ High reproducibility
■ Used at high pressures
● Sample inlet
● Ionization source
● Mass analyzer
○ Actual measuring of your mz ratio occurs when
the gas phase ions pass into
○ It will generate into an electrical field that can
manipulate the charge molecules to sort them
now according to mass ratios
● Ion detector
Electron ionization
- A method that requires a source of electrons into form a
filament to which an electric potential is being applied.
MAJOR STEPS:
● conversion of the parent molecule into a stream of ions
(usually singly charged positive ions)
● acceleration of ions in a magnetic or electrical field
● separation of the ions by mass/charge ratio (m/z)
● counting of the number of ions of each type or
measurement of current produced when the ions strike a
transducer
6
TWO TYPES:
Quadrupole mass spectrometer
● direct electrical current and radiofrequency voltages of

selected magnitudes are applied to two pairs of metallic
rods
● Only ions of specific mass/charge ratio can pass
undeflected to the end of the rods, where they are
detected
● Separation is based on the mass charge ratio; if it's not
the analyte that u are looking for then it is being deflected
into different direction and the only thing that could pass
thru is your analyte of interest.
Iron Trap Mass Spectrometer
● Three electrodes, in a ring shape and two end caps,

produce ions in the cavity until selectively ejected to the
ion detector
● ability to get full mass spectra at very low sample
concentrations
● Trapping electrons using electrodes.
TANDEM MASS SPECTROMETRY
● GC/MS/MS and LC/MS/MS

○ Common used in drug testing
○ Tandem - 2 mass spectrometry ang present
● used for greater selectivity and lower detection limits
● link three quadrupoles in series – triple quad
○ Q1 – used to scan across a preset m/z range
and select an ion of interest
○ Q2 – functions as a collision cell
○ Q3 – serves to analyze the product ions
generated in Q2 (full product ion scan or
selected reaction monitoring)

CC Lec - Prelim Exam

Uploaded by

Copyright:

Available Formats

CC Lec - Prelim Exam

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

CC Lec - Prelim Exam

Uploaded by

Copyright:

Available Formats

CLINICAL CHEMISTRY LEC

LECTURE 1: LABORATORY SAFETY AND

● Example on how we plan and ensure safety to the

Examples of Engineering Controls Administrative Controls

HAZARDS CLASSIFICATION: TYPES OF HAZARDS

Safety Hazards General Classification

POSTURE AND POSITION

LECTURE 2: EXPRESSING CONCENTRATIONS

Which among the 4 is very concentrated and the least 𝑥 (𝑚𝐿)

𝑣𝑜𝑙𝑢𝑚𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 𝑥 (𝑔)

𝑤𝑒𝑖𝑔ℎ𝑡 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒

Volume of solution= volume of solute + volume of solvent

Addressing the first question

𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒

● The final answer should be in a sentence form

𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒

Calculate the barium chloride concentration in mEq/L of a

How many grams of NaCl would be in a 100g of a 1 molal

𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒 𝑚𝑤 137.33 + 35.45 (2)

What is the normality of a solution that contains 150 grams

𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒

How do you make 500 mL of a 2N NaCl?

𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒

Example: 250 𝑚𝑔 10 𝑑𝐿 𝑔 𝑚𝑜𝑙𝑒 1𝑒𝑞 1000 𝑚𝑒𝑞

% = g/100mL 𝑚𝑔 𝑑𝐿 𝑔 𝑚𝑜𝑙𝑒 𝑒𝑞 𝑚𝑒𝑞

10 𝑚𝑔 10 𝑑𝐿 𝑔 𝑚𝑜𝑙𝑒 1000 𝑚𝑚𝑜𝑙

LECTURE 4: OPTICAL TECHNIQUES

● Can be described as discrete packets of energy

Important things to know:

● It means that it would take ½ second for a cycle to occur

● Wavelengths ranging from 1mm to 1m

● Different textbooks will give different wavelengths

Visible Light (Red)

Visible Light (Orange)

Visible Light (Yellow)

Visible Light (Green)

● What your eyes perceive, the thing absorbs

waves are in-phase

waves are out-of-phase

Pic left: true interference pattern of white light.

● If the concentration of the solution is low, and when u

● Glucose with Dinitrosalicylic acid (DNS)

○ Photodetector at the end

● Spectrum of visible light

Entrance and Exit Slits

Bandpass or Bandpass width

● Prism is the simplest monochromator

II. Interference Filters

● The polychromatic light is coming from this direction, but

● For blue wavelengths, once it is reflected back, it is

● analytical cell or sample holder

● Converts transmitted light energy into an equivalent

Barrier-Layer Cell (Photocell)

● Depletion Or intrinsic region

● How do we know that spectrophotometer is really giving

● used for Group 1 metals (1+ charge)

● Na+ – emits yellow light (589 nm)

ATOMIC ABSORPTION SPECTOPHOTOMETRY

LUMINESCENCE both electrons get combined, like a magnet = triplet state