Introduction to Bioseparation

Bioprocess Technology
 Introduction to Bioseparation

ü Modern biotechnology is built on the genetic manipulation

of organisms, genetic engineering, to produce commercial
products or processes.

ü Bioproducts have unique properties.

ü stability, solubilities, charge, isoelectric pH

ü Temperature, pH, and concentration must be maintained

within specific ranges to assure product bioactivity.
 Desired Product in Culture Broth

Intracellular product Extracellular product

(flocculation, filtration,
Recovery of cells centrifugation)
Removal of debris
(Physical, Chemical
Cell Disruption and enzymatic
method)
(Liquid-liquid extraction,
Removal of debris Concentration of extract membrane filtration,
precipitation, evaporation)

Purification by Chromatography (Gel-filtration, ion-exchange,

affinity. etc)

Formulation (Drying, freeze-drying,

crystallization)

Final Product
 Desired Product in Culture Broth

Intracellular product Extracellular product

1
(flocculation,
Recovery of cells filtration,
centrifugation) Removal of debris

2
(Physical,
Cell Disruption Chemical and
enzymatic
method) (Liquid-liquid

Removal of debris Concentration of extract

extraction,
membrane filtration, 3
precipitation,
evaporation)

Purification by Chromatography (Gel-filtration,

ion-exchange,
4
affinity. etc)

Formulation (Drying, freeze-

drying,
5
crystallization)

Final Product
 Stages of Downstream Processing

Downstream processing includes five different stages.

The five stages are:

(1) Solid-Liquid Separation

(2) Release of Intracellular Products
(3) Concentration
(4) Purification by Chromatography and
(5) Formulation.
Stage # 1. Solid-Liquid Separation:

The first step in product recovery is the

separation of whole cells (cell biomass)
and other insoluble ingredients from the
culture broth.

Several methods are in use for solid-liquid

separation. These include flocculation,
filtration and centrifugation.
1. Flocculation:

In flocculation, the cells (or cell debris)

form large aggregates to settle down for
easy removal. The process of flocculation
depends on the nature of cells and the
ionic constituents of the medium.

Example of flocculating agents :

inorganic salt, organic polyelectrolyte
2. Filtration:

Filtration is the most commonly used technique for

separating the biomass and culture filtrate.

Some of the examples of filters are depth filters,

absolute filters, and membrane filters.
2. Filtration:

The efficiency of filtration depends on many

factors—
ü the size of the organism,
ü presence of other organisms,
ü viscosity of the medium, and
ü temperature.
3. Centrifugation:

The technique of centrifugation is based on the

principle of density differences between the
particles to be separated and the medium.

Centrifugation is mostly used for separating solid

particles from liquid phase.
Stage # 2. Release of Intracellular Products:

The microorganisms or other cells can be

disintegrated or disrupted by physical, chemical or
enzymatic methods.
 CELL DISRUPTION BY PHYSICAL METHODS

The microorganisms or cells can be disrupted by certain physical methods

to release the intracellular products.

1. Ultra sonication:
Ultrasonic disintegration is widely employed in the laboratory. However, due
to high cost, it is not suitable for large-scale use in industries.

2. Osmotic shock:
This method involves the suspension of cells in 20% buffered sucrose. The
cells are then transferred to water at about 4°C.
 CELL DISRUPTION BY PHYSICAL METHODS

The microorganisms or cells can be disrupted by certain physical

methods to release the intracellular products.

3. Heat shock (thermolysis):

Breakage of cells by subjecting them to heat is relatively easy and
cheap. But this technique can be used only for a very few heat-stable
intracellular products.

4. High pressure homogenization:

This technique involves forcing of cell suspension at high pressure
through a very narrow orifice to come out to atmospheric pressure. This
sudden release of high pressure creates a liquid shear that can break
the cells.
 CELL DISRUPTION BY PHYSICAL METHODS

5. Impingement:

In this procedure, a stream of suspended cells at high velocity and pressure

are forced to hit either a stationary surface or a second stream of suspended
cells. The cells are disrupted by the forces created at the point of contact.

6. Grinding with glass beads:

The cells mixed with glass beads are subjected to a very high speed in a
reaction vessel. The cells break as they are forced against the wall of the
vessel by the beads. Several factors influence the cell breakage-size and
quantity of the glass beads, concentration and age of cells, temperature
and agitator speed.
 CHEMICAL METHODS OF CELL DISRUPTION

Treatment with alkalies, organic solvents and detergents can lyse the cells to
release the contents.

1. Alkalies:
Alkali treatment has been used for the extraction of some bacterial proteins.
However, the alkali stability of the desired product is very crucial for the success
of this method.

2. Organic solvents:
Several water miscible organic solvents can be used to disrupt the cells e.g.,
methanol, ethanol, isopropanol, butanol. The organic solvent toluene is
frequently used as it dissolves membrane phospholipids and creates membrane
pores for release of intracellular contents.
 CHEMICAL METHODS OF CELL DISRUPTION

3. Detergents:
Detergents : anionic-sodium lauryl sulfate (SDS) can denature
membrane proteins and lyse the cells.

Non-ionic detergents (although less reactive than ionic ones) are also
used to some extent e.g., Triton X-100 or Tween.
 ENZYMATIC METHODS OF CELL DISRUPTION:

Lysozyme is the most frequently used enzyme and is

commercially available (produced from hen egg
white).

As the cell wall gets digested by lysozyme, the

osmotic effects break the periplasmic membrane to
release the intracellular contents.

Certain other enzymes are also used, such as

glucanase, mannanase and proteases.
Stage # 3. Concentration:

The commonly used techniques for concentrating biological

products are evaporation, liquid-liquid extraction, membrane
filtration, precipitation and adsorption.

1. Evaporation:
Water in the broth filtrate can be removed by a simple evaporation
process.

2. Liquid-Liquid Extraction:
The concentration of biological products can be achieved by
transferring the desired product (solute) from one liquid phase to
another liquid phase, a phenomenon referred to as liquid-liquid
extraction.
3. Membrane Filtration:

The membrane filtration technique basically involves the use of a

semipermeable membrane that selectively retains the
particles/molecules that are bigger than the pore size while the
smaller molecules pass through the membrane pores.

4. Precipitation:

Neutral salts, organic solvents, high molecular weight polymers (ionic

or non-ionic), besides alteration in temperature and pH are used in
precipitation. The most commonly used salt is ammonium sulfate,
since it is highly soluble, nontoxic to proteins and low-priced.
Stage # 4. Purification by Chromatography:

The biological products of fermentation (proteins, pharmaceuticals,

diagnostic compounds and research materials) are very effectively
purified by chromatography.

Chromatography usually consists of a stationary phase and mobile

phase.

The stationary phase is the porous solid matrix packed in a column on to

which the mixture of compounds to be separated is loaded.

The compounds are eluted by a mobile phase.

Stage # 4. Purification by Chromatography:

The different types of chromatography techniques used for

separation :

Gel-filtration chromatography: size, shape and molecular weight

Ion-exchange chromatography: Net charge
Affinity chromatography: Biological affinity and molecular
recognition
Stage # 5. Formulation:

Formulation broadly refers to the maintenance of activity and

stability of a biotechnological products during storage and
distribution.

The formulation of low molecular weight products (solvents,

organic acids) can be achieved by concentrating them with
removal of most of the water.

For certain small molecules, (antibiotics, citric acid), formulation

can be done by crystallization by adding salts.
Stage # 5. Formulation:

Certain stabilizing additives are added to prolong the shelf life of

protein. The stabilizers of protein formulation include sugars
(sucrose, lactose), salts (sodium chloride, ammonium sulfate),
polymers (polyethylene glycol), DMSO and polyhydric alcohols
(glycerol).

Proteins may be formulated in the form of solutions, suspensions

or dry powders.

Dry powder can be achieved by drying, spray drying or freeze-

drying.
Chromatography

Physical method for separation of compounds in a sample.

Tswett- The father of chromatography

Chroma- color and graphein- writing

Plant pigments –chlorophyll xanthophyll

in calcium carbonate column
 Chromatography
separation

Stationary phase Mobile phase

 Sample / Solute / Analyte
interact

Stationary phase Mobile phase

• Solid • Liquid

• Liquid on solid • Gas

Chromatography

Separation is based on relative affinity to stationary phase vs mobile phase.

Analyte: The sample being analysed

Elution: The process of passing the analyte through stationary phase by

mobile phase

Eluent: The solvent used to pass the sample through stationary phase
i.e. mobile phase

Eluate: The solvent that exists the column

Chromatography: the separation technique

Chromatograph: The system used for chromatography

Chromatogram: The result profile of separation i.e intensity vs elution

volume/time
 Chromatographic methods

Shape of Physical state of Mechanism of

chromatographic bed mobile and stationary separation
phase
Shape of Chromatographic bed

Planar Column
chromatography chromatography
Shape of Chromatographic bed

Planar Column
chromatography chromatography

• Stationary Phase • Stationary Phase

on Paper in a tube/column
• Stationary Phase
on Plate- silica or • Mobile phase: Both
alumina gel on liquid and gas
glass/metal/plastic
plate

• Open bed
• Mobile phase:
Only liquid
Physical state of mobile phase

Gas Liquid
chromatography chromatography

Physical state of stationary phase

Gas-solid Liquid-solid
chromatography chromatography

Gas-liquid Liquid-liquid
chromatography chromatography
 Chromatographic methods

Shape of Physical state of Mechanism of

chromatographic bed mobile and stationary separation
phase
Planar

Column
 Chromatographic methods

Shape of Physical state of Mechanism of

chromatographic bed mobile and stationary separation
phase

Liquid/ Gas

Liquid-Liquid or Liquid-solid

Gas-Liquid or Gas-solid
 Chromatographic methods

Shape of Physical state of Mechanism of

chromatographic bed mobile and stationary separation
phase

Partition
Size-exclusion
Ion exchange
Affinity chromatography
Partition chromatography

Components get distributed between two phases

Based on respective solubilities in the two phases

Due to differences in partition coefficients (𝑲𝒅)

𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝒔𝒐𝒍𝒖𝒕𝒆 𝒊𝒏 𝒑𝒉𝒂𝒔𝒆 𝑨

𝑲𝒅 =
𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒐𝒇 𝒔𝒐𝒍𝒖𝒕𝒆 𝒊𝒏 𝒑𝒉𝒂𝒔𝒆 𝑩
 Liquid-Liquid Partition chromatography

Normal-phase partition chromatography: Reversed-phase partition chromatography:

Stationary phase >> Mobile phase Mobile phase >> stationary phase

Most non-polar solute elutes first Most polar solute elutes first

Most polar solute elutes last Most non-polar solute elutes last
Paper chromatography

Type of partition chromatography

Paper dipped in solvent mixture

containing both

Aqueous components Organic components

Binds to cellulose of paper- stationary phase Keeps migrating- mobile phase

Polar analyte will migrate slower as compared to non-polar

Paper chromatography

Migration of the analyte based on relative solubilites and partition coefficient

𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒕𝒓𝒂𝒗𝒆𝒍𝒍𝒆𝒅 𝒃𝒚 𝒔𝒖𝒃𝒔𝒕𝒂𝒏𝒄𝒆

𝑹𝒇 =
𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒕𝒓𝒂𝒗𝒆𝒍𝒍𝒆𝒅 𝒃𝒚 𝒔𝒐𝒍𝒗𝒆𝒏𝒕 𝒇𝒓𝒐𝒏𝒕
Two- dimensional paper chromatography

Enhance the separation of the mixture into its components.

 Thin-layer chromatography
The separation principle of the TLC procedure is based on
the given compound’s relative affinity towards the mobile
and the stationary phase. The higher affinity compounds
gain less speed as compared to the lower affinity
compounds. TLC is used to separate non-volatile mixtures.
Uses of Thin-layer chromatography (TLC)

1. Thin-layer chromatography is routinely performed in

laboratories to identify different substances present in
a mixture.
2. This technique helps in the analysis of fibers in
forensics.
3. TLC also allows the assay of various pharmaceutical
products.
4. It aids in the identification of medicinal plants and their
composition.
Size-exclusion chromatography

Separation is based on shape and size

Column is filled with porous gel beads

Polyacrylamide (Sephacryl) or dextran (Sephadex)
or agarose (Sepharose)

Dextran: glucose polymer produced by the bacterium

Leuconostoc mesenteroides,
Agarose: polymer of alternating D-galactose
and 3,6-anhydro-L-galactose from red algae,

Large molecules cannot enter bead pores, elute first

Small molecules enter bead pores, longer retention time,
elute later
Size-exclusion chromatography
Size exclusion limit

The smallest molecule in size that is not able to enter the beads.

Sephadex G-50 has exclusion limit of 30 kDa

Molecules> 30kDa pass directly

Material Column Fractionation range (kDa)

Dextran Sephadex G-100 4-150
Sephadex G-150 5-300
Agarose Sepharose 2B 2000-25000
Sepharose 4B 300-3000
Polyacrylamide Bio-gel P-6 1-6
Bio-gel P-60 3-60
Column Characteristics

𝑽𝑶 + 𝑽𝑿 = 𝑽𝑻

𝑽𝑶 external volume surrounding the beads

𝑽𝑿 volume occupied by beads
𝑽𝑻 total bed volume

𝑽𝑶 is typically 35% of 𝑽𝑻.

Elution volume 𝑽𝒆
The solvent volume required to elute the solute from the
column.

Relative Elution volume

For a particular solute : defined by ratio 𝑉! / 𝑉" ,
Independent of the column used.
Ion exchange chromatography

Separation of charged molecules

Charged molecules show electrostatic interactions with charged stationary matrix

Charged stationary matrix/ Ion exchangers= Anions/cations covalently bonded to matrix

Diethylaminoethyl (DEAE)-cellulose
Weakly basic- Anion exchanger-
used for anion separation

Carboxymethyl (CM)-cellulose
Weakly acidic- Cation exchanger
used for cation separation
Ion exchange chromatography

Anion exchanger- used for anion separation

Name Type Functional group

DEAE-cellulose Weakly basic Diethylaminoethyl (DEAE)
QAE-Sephadex Strongly basic Quarternary aminoethyl (QAE)
Q-Sepharose Strongly basic Quarternary ammonium (Q)

Cation exchanger- used for cation separation

Name Type Functional group

CM-cellulose Weakly acidic Carboxymethyl (CM)
SP-Sepharose Strongly acidic Sulfopropyl (SP)
SOURCE S Strongly acidic Methylsulfate (S)
Ion exchange chromatography

Analyte – depending on the charge

Bind with the ion exchanger

Elution is done by solvent of increased ionic strength or pH to release the bound analyte by
displacement

Stepwise elution
Ion exchange chromatography
Selection of stationary and mobile phase

Amino acid
Selection of stationary and mobile phase

pH and ionic strength

Stability of the analyte is maintained
Should not prevent the interaction of analyte with matrix at first

Stepwise elution
Ion exchange chromatography

Can also be used to separate DNA

Negatively charged phosphate of DNA backbone interact with anion exchanger

DEAE on silica beads

Eluted using salt solution of higher concentration

Affinity chromatography

Separation based on biological function/chemical structure/ability to bind specific molecules

Ligand is covalently attached to inert and porous matrix stationary phase

Under favorable conditions, specific binding of desired protein from mixture occurs with
ligand

Unbound proteins wash off

Desired protein can be eluted by changing conditions to release the binding from matrix
Affinity chromatography
Affinity chromatography

Advantage

Exploits the unique properties of the desired protein rather than the small
differences in physicochemical properties
Affinity chromatography

Choice of appropriate ligand

Matrix for immobilization of ligand

Binding of desired molecule with ligand

Removal of contaminating molecules

Elution of molecules of interest

 Affinity chromatography

Choice of appropriate ligand

Specificity

Recognize and bind only the molecule of interest to be purified

Choice of appropriate ligand

Ligand Target molecule

Enzyme Substrate or inhibitor or cofactor
Antibody Antigen
Nucleic acid Complementary sequence, binding protein
Avidin Biotin
Poly (A) Poly U or poly T
Glutathione Glutathione-S-transferase or GST-fusion protein
Proteins A and G Immunoglobulins
 Affinity chromatography

Choice of appropriate ligand

Specificity Reversibility

Reversible complex between

ligand and molecule of interest
 Affinity chromatography

Choice of appropriate ligand

Specificity Reversibility Stability

Ligand should be stable –

immobilization and
chromatography conditions
 Affinity chromatography

Choice of appropriate ligand

Specificity Reversibility Stability Size

Appropriate size with

fairly enough interacting
groups
 Affinity chromatography

Choice of appropriate ligand

Specificity Reversibility Stability Size Affinity

Affinity chromatography

Choice of appropriate ligand

Matrix for immobilization of ligand

Binding of desired molecule with ligand

Removal of contaminating molecules

Elution of molecules of interest

Matrix for immobilization of ligand

Chemically inert,

Have high porosity,

And have large numbers of functional groups capable of forming covalent

linkages to ligands.

polymer of alternating D-galactose

and 3,6-anhydro-L-galactose from red algae,
Affinity chromatography

Protein purification
Affinity chromatography

mRNA purification
Affinity chromatography Purification of DNA-binding proteins
Affinity chromatography Purification of DNA-binding proteins
Uses of Affinity chromatography

1. Affinity chromatography is used as a staple separation

technique from enzymes and other proteins.
2. This technique is used for the separation of components
as well as the removal of impurities from a mixture.
3. Affinity chromatography can be used in the detection of
mutation and nucleotide polymorphisms in nucleic acids.
 Gas chromatography
Gas chromatography is a separation technique in which the
molecules are separated on the basis of their retention time
depending on the affinity of the molecules to the stationary
phase. The mobile phase is a gas, mostly helium, that carries
the sample through the column.
Uses of Gas chromatography

1. This technique is used to calculate the concentration

of different chemicals in various samples.
2. This is used in the analysis of air pollutants, oil spills,
and other samples.
3. Gas chromatography can also be used in forensic
science to identify and quantify various biological
samples found in the crime scene.
Types of chromatography

Analytical vs Preparative

Analyzing Preparing
Separation of components from Separation, purification and
mixture isolation of a particular
component from mixture
Small scale Large scale
Liquid Chromatography
Ø The solvents should be highly purified

Ø Degassing methods
FPLC

Ø Fast protein liquid chromatography (FPLC) is a form of

medium-pressure chromatography

Ø pump to control the speed at which the mobile phase passes

through the stationary phase.

Ø introduced in 1982 by Pharmacia as fast performance liquid

chromatography.
FPLC

Ø The aim of the FPLC user is to obtain as much pure and

native product as possible.

Ø The working temperature for proteins is usually 4 °C.

Ø FPLC machine is often placed inside a cold chamber or cold

room
HPLC

Ø High performance or high-pressure liquid chromatography

(or) HPLC

Ø Small particle size of stationary phase and the use of high

pressure

Ø separation of biomolecules with high performance

parameters under high pressure by using appropriate
instrumentation.
HPLC

Ø Analyze small chemical compounds

Ø The aims of classical HPLC are to identify and qualify

analytes, usually small compounds

Ø In a medical setting : determine the contents and

concentrations of substances in biological materials. For eg:
drug analysis of urine or detection of vitamin levels in blood
serum

Ø Detection of impurities in pharmaceutical industries

HPLC

Ø pressure-resistant stainless steel columns are used

Ø stationary phase particles are rigid rather than soft gel as in

open column chromatography.

Ø The particles are spherical and in uniform size to reduce

space for diffusion.
UPLC

Ø Ultra performance liquid chromatography

Ø Decreasing the particle size and increasing the capacity of

instruments

Ø Stationary phase particle size of 1.7 μm diameter, made up of

Bridged Ethylsilixane Silica Hybrid (BEH).

Ø This chromatography is 10 times faster than conventional

HPLC.
Advantages of UPLC

Shortening analysis time up to nine times.

Provides the selectivity, sensitivity, and dynamic range of LC analysis.

Maintains resolution performance.

Fast resolving power quickly quantifies related and unrelated

compounds.

Separation on UPLC is performed at very high pressures up to 15000psi.

Introduction to Bioseparation
Process and Product Quality

The measures of product quality due to processing are :

ü purity,

ü fold purification,

ü specific activity, and

ü yield.
Process and Product Quality

Purity
Process and Product Quality

Fold purification is the ratio of the purity at any stage in

the process to the purity at the start of the purification
process.

𝑥
𝑦
Process and Product Quality
Process and Product Quality
Process and Product Quality

Strictly quantitative measure and not always an

expression of the quality of the product.
Process and Product Quality

Therapeutic protein can be 99.99% pure but still unacceptable

if any pyrogen (a substance that produces a fever) is present.

Industrial enzyme, then practically any impurities that do not

inhibit the activity of the product or endanger the user are
allowed.
Process and Product Quality
Criteria for Process Development

Approaches to process development (also known as

process synthesis) must be made with future scale-up in
mind.

Product production process is a set of connected unit

operations that produces a final product able to meet
defined levels of quality, yield, and cost.
Criteria for Process Development

Evaluating and developing a bioseparation process:

• Product purity

• Cost of production as related to yield

• Scalability

•Reproducibility and ease of implementation

Interdisciplinary task, involving not only engineers and bioscientists but also marketing
and regulatory personnel.
ü Erythropoietin (a peptide hormone that
stimulates red cell production in the bone
marrow)
ü Oligonucleotides,
Analytical methods during
Downstream Processing
Bioprocess Technology
ü Efficient and reliable processes

ü Analytical methodology for the bioproduct: beginning of

process development

ü Changes can occur either to the product or to its

associated impurities
ü Identify “specifications” that are desired, especially by
regulatory agencies, in the definition of a product.

ü Define assay attributes in terms of precision, accuracy,

specificity, linearity, limits of detection, range, and
robustness.

ü Identify and select laboratory methods for assaying

biological activity.

ü Identify and select laboratory methods for assaying purity.

ü Define measurements made in microbiological assays.

 Specifications

ü Set for any product that is expected to be sold

ü For assuring the quality and consistency of the product

ü Pharmaceutical: U.S. Food and Drug Administration

specifications

ü Manufacturer assures through routine measurements such

product attributes as biological activity, purity, identity,
physical characteristics (including color and other
appearance qualities), and product safety.

ü Central Drugs Standard Control Organisation(CDSCO) &

Indian Pharmacopoeia Commission
 Specifications

ü Identity determination (HPLC, peptide map, sequencing)

ü Biological activity (bioassay: specific activity)

ü Purity [ultraviolet, reversed-phase HPLC, total and individual

related substances, multimeric forms, residual organic
solvents by gas chromatography, trace metals, host-cell
proteins, endotoxin, moisture, particulates, and sterility]

ü Physical/appearance qualities (general inspection, physical

appearance, solubility, pH, and content uniformity)
Specifications
Specifications
 Specifications

ü The specification usually evolves as the process is

developed and the product’s properties are learned.

ü It is always best to start with broad specifications that are

narrowed as the product is developed for market.

ü Safety specifications are of the utmost importance and

must be well thought out from the onset of process
development.
Assay attributes
 Assay and its attributes

ü The effectiveness of a method for measuring attributes

depends on its

ü precision,
ü accuracy,
ü specificity,
ü linearity,
ü range, and
ü robustness.
 1. Precision

ü Measure of the reproducibility of an assay

ü Tells the likelihood that a repeat test will give the same
result.

ü Precision is expressed as a relative standard deviation

(RSD), also known as the coefficient of variation (CV),
defined as the standard deviation divided by the
average.

ü Converted to a percentage

ü Assays for which the RSD is greater than 5% are

generally unacceptable
 Precision

ü Perform replicate analyses on a reference standard or other

well-characterized material.

ü Ideally, all external factors that can add to the variability of

the assay are minimized or reduced.

ü The sample and all reagents : should have good stability and
should give a expected result
Precision
 Precision

ü Estimate of the probable error of a single measurement.

ü At least three replicates must be made before standard

deviation can be determined

ü The standard deviation of the mean is usually referred to as the

standard error.
 2. Accuracy

ü Measure of the closeness of the assay result to the “true

value.”

ü Spiking the sample with a known amount of standard allows

the analyst to determine the amount of an unknown quantity
that is likely to be measured by the assay.

ü The spiked standard should be of known content and purity,

because the exact quantity of standard introduced into the
sample matrix provides the basis for the assay.

ü Accuracies within 1% are exceptional; assays outside 5% are

typically unacceptable.
 Accuracy

ü Accuracy is more dependent on the sample preparation

and storage conditions than on the analytical method itself.

ü Variety of different materials, or pretreatments for materials,

may be used to increase accuracy.
 3. Specificity

ü Distinguish between the analyte and similar components

ü Specificity always means that no other molecules in the

sample matrix interfere with the quantitation of the target
molecules

ü An assay designed to quantitatively resolve two related

chemical impurities should not also quantitate DNA
 Specificity

ü Estimation of a protein product by HPLC: other deletion or

misincorporation products, any such contaminants must be
sufficiently resolved from the product to be accurately
quantitated

ü No single HPLC technique is specific for all possible product-

related impurities

ü For this reason, HPLC techniques are almost always used in pairs
called orthogonal methods, so that a broader range of
contaminants can be detected.
 Specificity

ü Identity- detection method, such as mass spectrometry (MS)

attached to an HPLC

ü tryptic (or other enzymatic) maps,

ü specific precipitation or colorimetric assays (such as the enzy-

matic cleavage of a specific substrate to a pigment product),

ü elemental analysis
4. Linearity, Limit of Detection, and Limit of Quantitation

ü Linearity: a response proportional to the concentration of the

analyte.

ü by creating a standard curve for the analyte(s)

ü Assays become nonlinear at both high and low concentrations,

where detectors become saturated (high) or nonspecific
responses or noise become a significant proportion of the
response (low).
4. Linearity, Limit of Detection, and Limit of Quantitation

ü When a standard curve is constructed via regression, a slope,

intercept, and r2 result.
4. Linearity, Limit of Detection, and Limit of Quantitation

ü The LOD is the lowest analyte concentration that can be

distinguished from the assay background,

ü LOQ is the lowest concentration at which the analyte can be

quantitated at defined levels for precision and accuracy

ü The noise inherent in the system may sometimes be assessed by

analysis of “blank” samples.

ü Sometimes it is specified by the instrument being used.

Frequently, LOD is three times the noise, and LOQ is ten times the
noise.
5. Range

ü Upper and lower limits within which the assay can produce
accurate and precise results.

ü Concentration or the amount of the analyte;

ü Solution properties: pH, temperature, and “matrix” composition

ü Assay range is reported in the units that describe the sample

attributes for which the assay is valid (e.g., pH 6.5–7.5)

ü Accuracy and precision of the result do not vary across the

assay range.
6. Robustness

ü “Robustness” refers to the assay conditions.

ü In an HPLC method, for instance, flow rate, temperature, pH

of the buffers, and solvent content of the buffers.

ü Critical parameters, or those that are inherently hard to

control, need to be examined.

ü Interacting factors will play a role, although the precise role is

rarely measured.