Chromatography Flashcards
Analytical Separation, General Principles of Chromatography, Gas Chromatography, Liquid Chromatography
Separation techniques based on the size (4)
- filtration
- dialysis
- size-exclusion chromatography
- electrophoresis
Separation techniques based on the mass or density (1)
centrifugation - spinning sample at high speed
Separation techniques based on complex formation (1)
masking/demasking
Separation techniques based on change in physical state/physical method (3)
- distillation
- sublimation
- crystallization
Separation techniques based on change in chemical state/chemical method (3)
- precipitation
- electrodeposition
- volatilization (involves chemical reaction)
Separation techniques based on partitioning between phases (2)
- extraction
- chromatography
separates a particular interferent from solute analytes using a filter with a pore size that will retain the interferent (particle size must be consider)
filtration
Differentiate filtrate and retentate
- Filtrate - solution that passes through the filter
- Retentate - material that is retained by the filter
used for particulates that are too small to be retained by filter paper
membrane filter
Differentiate types of membrane filter (3)
- centrifugal filter - macromolecules (10^6 g/mol)
- syringe filter - smaller particles (0.45 µm)
- disposable filter - cellulose acetate (0.22 µm)
binding the interferent in a strong, soluble complex that prevents it from interfering in the analyte’s determination
masking
process of separating two liquids that have different boiling points.
- liquid is converted to its vapor
- collecting the vapor as liquid by condensation
distillation
Differentiate types of distillation (4)
- Simple distillation
- analyte does not go decomposition before its boiling point (large boiling point difference) - Fractional distillation
- similar boiling points; uses fractionating column - Vacuum distillation
- substances that boil above 200 ºC at 1 atm
- distillation at reduced pressure - Steam distillation
- passing dry steam through the sample whereby the steam volatile compounds are volatilized
process of collecting the distillate in several fractions and subjecting the fractions to systematic redistillation
rectification
type of short-path vacuum distillation.
used to separate substances that decompose at the boiling temperature even in high vacuum
molecular distillation
Used if the material to be extracted is
immiscible, chemically non-reactive with water, and temperature sensitive
Steam Distillation
Describe sublimation and when it is used?
a solid directly converted into gas without converting into the liquid phase
- mostly used for separation of non-volatile compounds from volatile compounds
compounds that are capable of sublimation tend to be those with _____ such as ___
weak intermolecular forces in the solid state
compounds with symmetrical or spherical structure
How to choose solvent to use in crystallization?
When solvent is hot - significant solubility of analyte/ less soluble interferant
When solvent is cold - minimal solubility of analyte/
Process of recrystallziation
- cycle of adding hot solvent and sample then cooling in ice bath
- rinsing and drying the crystals
- recrystallize for further purification
converting the analyte in another form before separating
Separation by chemical reactivity
require large solubility differences between the analyte and potential interferences
Separation by precipitation
____ are good reagent for separating metal since most metal ions except alkaline and alkali are _____
Sulfides; very insoluble with sulfides
_____ (3) ions are often used as precipitants for cations, but they are not ____
phosphate, carbonate, and oxalate
selective
chloride can separate ____ from ____
silver; most other metals
sulfate can isolate a group of metals that includes _____
lead, barium, and strontium
Separate nickel ion using ____ precipitant
dimethylglyoxime
Separate aluminum ion using ____ precipitant
8-hydroxyquinoline
metals are deposited in an electrode by controlling the potential of the working electrode
Separation by Electrolytic Precipitation
How will you separate a miscible solution of benzene and CHCl3?
Distillation - separation of two liquids
Iodine can be purified by ____. Why?
Sublimation, iodine is volatile and can sublime
A mixture of camphor and KCl is best separated by ____
sublimation - camphor can be easily sublime
A mixture of camphor (BP = 209 degree C) and benzoic acid (BP = 249 degree C) can be separated by ____. Why?
Chemical method
- benzoic acid, an acid can react with the base, but camphor is an organic compound which does not react with either acid or base, hence, NaHCO3 will be used to react with benzoic acid which will become sodium benzoic which is water soluble that can be extracted
o-Xylene and m-Xylene products of the same compounds can be separated by ____
crystallization - different melting point
A mixture of iron and copper filings can
be separated by ____
physical method (magnet separation0
the transfer of a solute from one phase to
another to isolate or concentrate the desired analyte
extraction
Types of extraction (4)
- liquid-solid (SPE)
- liquid sample
- solid extractant (absorbent) - solid-liquid
- solid sample
- liquid extractant - liquid-gas
- liquid sample
- gas extractant - liquid-liquid
- liquid sample
liquid extractant
Types of Adsorption Solid Phase Extraction (SPE)
- Normal - least polar compounds elute first
- Reverse - most polar compounds elute first
Types of Ion Exchange Solid Phase Extraction (SPE)
- Cation - Weakly ionized compounds elute first; cations retained
- Anion - Weakly ionized compounds elute first; anions retained
In solid-liquid extraction, continuous extraction of a solid sample is carried out using a ____
Soxhlet extractor
Solid-liquid extraction is used when ____
used when the desired compound has a limited solubility in a solvent and the impurity is insoluble in that solvent
Three main sections of solid-liquid extraction
- Percolator (boiler and reflux)
- circulates solvent - Thimble
- retains the solid to be extracted (made of thick filter paper or membrane) - Siphon mechanism
- periodically empties the thimble
In liquid-gas extraction, _____ can be quantitatively removed from a liquid sample by a method called _______
volatile organic compounds (VOCs)
purge-and-trap
Explain liquid-liquid extraction and its phases
the solute partitions itself between two immiscible phases which usually done using separatory funnel
- Aqueous solvent
- Organic solvent
_____ (3) are common solvents that are immiscible with and less dense than water (floats on top)
Diethyl ether, toluene, and hexane
_____ (3) are common solvents that are denser than water (below aqueous phase)
Chloroform, dichloromethane, and carbon tetrachloride
Many _____ extractions are more effective than a few ____ extractions
Many small extractions are more effective than a few large extractions
If a solute is an acid or base, its charge changes as the pH is changed. Usually, a ____ is more soluble in an organic solvent and ____ is more soluble in aqueous solution.
neutral species is more soluble in an organic solvent and a charged species is more soluble in aqueous solution.
The _____ is used in place of the partition coefficient K when dealing with a species that has more than one chemical form
distribution coefficient D
For an acidic solute, the extraction efficiency is greater at ____ pH levels because _____
more acidic
HA is the solute’s predominate form in the aqueous phase
The extraction efficiency is independent of pH for pH levels _____ and essentially zero for pH levels ____
independent = more acidic than the HA’s pKa
~zero = more basic than HA’s pKa
the greatest change in extraction efficiency occurs at pH levels where _____
both HA and A – are predominant species
Chromatography derived from Greek word ____ and ____
chroma means “color”
graphein means “to write”
Chromatography is a separation technique based on the ____ as the compounds travels through a supporting medium
different interactions of compounds with two phases, a mobile phase and a stationary phase
General components of chromatography (4)
Mobile Phase: a solvent that flows through the supporting medium
Stationary Phase: a layer or coating on the supporting medium that interacts with the analytes
Column: contains the stationary phase, allowing the mobile phase to pass through it
Supporting Medium: solid surface on which the stationary phase is bound or coated
Eluent ___ → Eluate ____
Define both terms
Eluent in → Eluate out
Eluent – fluid entering a column
Eluate – fluid exiting the column
the process of passing the mobile phase through the column
Elution
solvent used in the elution
Eluant
graph showing detector response as a function of a time
chromatogram
invented chromatography in 1906 during his research to separate plant pigments like chlorophylls and carotenoids
- aka father of chromatography
Mikhael Tswett
In Column Chromatography,
○ _____ – adsorbent
○ _____ – eluent
calcium carbonate – adsorbent
ether/ethanol mixture – eluent
___ Nobel prizes were awarded between 1937 and 1972 alone for work in which chromatography played a vital role.
12
The primary division of chromatographic techniques is based on the _____ in the system
type of mobile phase used
Two theories explain the phenomenon in chromatography
Rate Theory: proposed by Van Deemter in 1956
Plate Theory: developed by Martin and Synge in 1941 (1952 Nobel Prize)
This theory assumes that the column is divided into a number of zones called theoretical plates
plate theory
‘imaginary’ measures how efficiency a column can separate a mixture into its components
theoretical plates (N)
To achieve efficient separation, theoretical plate (N) must be ____ and the plate height or Height Equivalent of a Theoretical Plate (H or HETP) ______
large N
small H
Describes the relationship between the height of a theoretical plate (H) and the linear velocity (u) of the mobile phase in chromatography
rate theory
Sources of Band Broadening (3)
- Multiple Path in Column
- Molecular Diffusion
- Mass Transfer
a band of molecules in a chromatographic peak in solvent or gas phase will _____ because of diffusion
gradually spread out
the faster we can get the analyte through the column, the ____ will the peak be broadened by the diffusion
less
increased ______ velocity increases contribution to spread by resistance to mass transfer
mobile phase
Explain the sum of effects of sources of band broadening
- Multiple path effect is independent of mobile phase velocity
- Diffusion effect is inversely proportional to the mobile phase velocity
- Mass transfer is proportional to the mobile phase velocity
sum of different variables will obtain _____
optimal velocity
the ____ of optimal velocity, the
ideal the mobile phase velocity
lowest value
a process that leads to peak (band) broadening due to the presence of multiple flow paths through a packed column
Eddy diffusion (uneven flow)
“A” variable in HETP formula
Eddy diffusion is ____ with mobile phase velocity, u
independent
Eddy diffusion (A) must be ____ to minimize or have a smaller plate height (HETP)
minimized
The degree of band-broadening due to Eddy diffusion and mobile phase mass transfer depends mainly on the (2)
- diffusion rate of solute
- size of packing material
band-broadening due to the diffusion of the solute along the length of the column in the flowing mobile phase
Longitudinal diffusion (molecular diffusion)
B variable in HETP equation
longitudinal diffusion (B) is ____ to flow rate (u)
inversely proportional
The degree of band-broadening due to longitudinal diffusion depends mainly on the (2):
- diffusion of solute
- flow rate of solute through column
band-broadening due to differences in the rate of diffusion of the solute molecules between the mobile phase outside the pores of the support (flowing mobile phase) to the mobile phase within the pores of the support (stagnant mobile phase)
Resistance to Mass Transfer (C)
resistance to mass transfer (C) is ____ with flow rate
directly proportional
The degree of band-broadening due to stagnant mobile phase mass transfer depends mainly on the: (3)
- flow rate of the solute through the column
- diffusion and retention of the solute
- size, shape, and pore structure of the packing materials
The degree of band-broadening due to stationary phase mass transfer depends mainly on the (3):
- flow rate of the solute through the column
- diffusion and retention of the solute
- kinetics of interaction between the solute and stationary phase
The effect of different variables giving the optical velocity is ____
Van Deemter plot
information that can be derived from Van Deemter plot is the ______ which has a minimum band-broadening and related to the plate height
ideal mobile phase velocity
smaller plate height, ______
more ideal mobile phase velocity
How to achieve optimum velocity? (2)
- Use smaller size packing material
- Use uniform size and shape
smaller particles will ____, limiting peak broadening by the multiple path effect (A-term)
give more similar paths
smaller particles lead to _____ and therefore reduce the C-term
more exchange between the phases
irregularly shaped particles create void volumes that are _____
not efficiently flushed by the mobile phase
The _____ is usually not of importance in LC
diffusion coefficient (B-term)
time that a solute spends in the stationary and mobile phases
Retention time
volume of the mobile phase required to carry the solute through the column to elution
(a measure of the fraction of time spent by
the solute in the mobile phase)
retention volume
Retention on a given column pertains to the particulars of that system: column size and flow rate of the mobile phase
solute retention
measure of the time the sample component resides in the stationary phase relative to the time it resides in the mobile phase
retention factor
(capacity factor)
retention factor is ____ on column length and flowrate and hence, useful for ____
independent
comparing results obtained on different systems
way of identifying how good the separation
retention factor
k’ < ____, separation is poor
k’ > ____, separation is slow
k’ is ____, separation is optimum
k’ < 1.0, separation is poor
k’ > 30, separation is slow
k’ is 2-10, separation is optimum
ability of the chromatographic system to ‘chemically’ distinguish between sample components
selectivity factor
(relative retention or separation factor)
selectivity factor is always _____
greater than 1
inverse of the ratio of the speeds at which the two components travel
unadjusted relative retention
The separation of solutes in chromatography depends on (2)
- difference in the retention of solutes
- sufficiently narrow width of the solute peaks
the farther apart the difference in the retention of solutes, the _____
better their separation
the wider the peaks, the _____
poorer their separation
How to improve separation? (2)
- retention factor (k’) is optimized first
- selectivity factor (α) is increased
How to increase selectivity factor (α)? (4)
- changing mobile phase composition
- changing column temperature
- changing composition of stationary phase
- using special chemical effects
(such as incorporating a species which complexes with one of the solutes into the stationary phase)
another measure of the separation of two peaks of different retention time
resolution
____ baseline resolution = ideal/good separation
1.5
____ retention, the more prolong/extended time
higher retention
____ selectivity, two peaks are far from each other
higher selectivity
____ efficiency, there is an overlap in two peaks
lower
How to know which column has more theoretical plates?
sharper peaks = more efficient separation
How to know which column has larger plate height (HETP)?
Broader peaks
large plate height = fewer plates
How to know which column gives higher resolution?
less overlap of peaks
How to know which compound has greater retention factor?
longer retention time
How to know which compound has greater partition coefficient?
longer retention time = greater affinity for stationary phase
How to know the numerical value of unadjusted relative retention?
retention time of component 2 / retention time of component 1
Gas-Solid Chrom = solid stationary phase
Gas-Liquid Chrom = ____
liquid stationary phase
Gas-solid chromatography uses ____ technique while Gas-Liquid Chromatography uses ____
Adsorption chromatography = GSC
Partition chromatography = GLC
Distribution coefficients of compounds are ____ in GSC while ____ in GLC
much higher = GSC
comparatively much smaller = GLC
retention time is ____ in GSC while ____ in GLC
comparatively long = GSC
comparatively short = GLC
Since liquid phase is absent in GSC, ____ can be used
higher temperature
Higher temperature in GLC is determined by the ____
nature of liquid phase
Comparatively very small concentration of samples can be used in ____ while higher concentration of samples can be used in ___
small conc = GSC
high conc = GLC
most commonly used to separate organic compounds
gas-liquid chromatography
for separating species not retained by gas-liquid columns
gas-solid chromatography
not much of ____ can be adsorbed on the stationary phase and can be easily removed
low molecular weight gases
Describe the instrumentation of Gas Chromatography (8)
- Carrier Gas Tank
- Pressure Regulator
- Flow Controller
Inside Column Oven:
4. Sample Injection Port
5. Column
6. Detector
- Processor Signal
- Outlet
inert gas used to carry samples
carrier gas
carrier gas should be high purity of at least ____. if not, ____ can occur
99.995%
interference can occur
first choice carrier gas that provides fastest separations due to low molecular weight
hydrogen
Disadvantages of hydrogen carrier gas
flammable and explosive
Safer but more expensive option for carrier gas
helium
Disadvantages of helium carrier gas
scarcity
carrier gas with smaller diffusion coefficient, slower, relatively higher column efficiency, safe, lower price
nitrogen
Disadvantages of nitrogen carrier gas
low thermal conductivity, poor sensitivity, narrow quantitative linear range, lesser resolution
should deliver gas with uniform pressure of flow rate
pressure regulatory or flow controller
placed before column inlet that has a glass tube with a float held on to a spring
rotameter
the level of the float of rotameter is determined by the ____
flow rate of carrier gas
soap bubble formed indicates the flow rate
soap bubble flow meter
rubber disk with self-sealing rubber
septum
happened when sample injection system reached its lifespan as observed in sharp repetitive peaks
septum bleed
Explain sandwich injection
Air, solvent, air, sample, air
air bubble behind the sample plug ____
prevents sample and solvent from mixing
Sample injection technique best for thermally unstable solutes and high-boiling solvents; best for quantitative analysis
On-Column Injection System
Sample injection routine for introducing small sample volume
split
Sample injection technique best for trace levels of high-boiling solutes in low-boiling solvents
Splitless
Sample injection that has glass insert whereas the entire sample can be injected into a packed column
direct injection
sample injected as is by inserting tip of the microsyringe needle directly into the tip of the capillary column, while maintaining injection port temperature below the boiling point of sample solvent (no glass insert)
on-column injection
injection technique suitable for samples with low concentrations of measurement components
on-column
Disadvantage of on-column injection
periodic maintenance is required (dirty column)
injection technique for the analysis of compounds that are thermally unstable and can be done using split or splitless injection
Programmed Temperature Vaporization (PTV) Injection
In PTV injection, when sample is injected, the injection port is set to _____ of the injection sample solvent
below the boiling point
after the sample is injected in PTV injection, injection port is _____, causing the injected sample to vaporize
heated rapidly
Injection technique used if expecting very high concentration of analyte where only a portion is injected into the column and hence, not suitable for trace analysis
Split injection
injection technique where most of the sample is injected, the remainder is eliminated
splitless injection
injection technique used for low-concentration samples that require higher sensitivity
splitless injection
injection technique NOT suitable for gas samples, low boiling point solvent samples, and components eluted in the vicinity of solvents
splitless injection
Compare the retention time, peak area, and peak width using split and splitless injection
Larger and broader retention time, peak area, and peak width in splitless injection
Sample introduction techniques (3)
- solid-phase microextraction
- headspace sampling
- purge-and-trap
If same polarity, the elution will be based on ____
order of increasing boiling point
Types of column (2)
- Packed Column
- Capillary Column/ Open Tubular
In _____, stationary phase is coated directly in the column while in _____ stationary phase is
coated with the inner wall of the column
Packed column - directly
Capillary column - inner wall
____ column is applicable only for GLC while ____ column is applicable for both GSC and GLC
Capillary Column - GLC
Packed Column - GSC and GLC
In ____, liquid stationary phase is immobilized on the capillary tubing walls while in _____, liquid stationary phase is adsorbed onto the surface of the beads in a thin layer or onto the solid inert packing
Capillary tube - liquid stationary phase is immobilized on the capillary tubing walls
Packed Column - liquid stationary phase is adsorbed onto the surface of the beads in a thin layer or onto the solid inert packing
____ column = 2-6 m length, 2-4mm id
____ column = 15-100 m length, 150-300 um id
Packed column = 2-6 m length, 2-4mm id
Capillary column = 15-100 m length, 150-300 um id
____ column = will require a higher flow rate (25 - 150 mm per min)
____ column = flow rate under 25 mm per min
Packed column = will require a higher flow rate (25 - 150 mm per min)
Capillary column = flow rate under 25 mm per min
made up of stainless steel or glass tube filled with particulate packing material (an adsorbent material, or a support material coated or impregnated with a solid phase)
Packed Column
solid support of packed column is often ___ that is ____ to reduce hydrogen bonding to polar solutes
silica that is silanized
compared with open tubular columns, packed columns provide ____ capacity but give ____, ______, and ______
greater sample
broader peaks, longer retention times, and less resolution
long narrow open tubular columns that are made of fused silica (SiO2) and coated with polyimide (a plastic capable of withstanding 350°C) for support and protection from atmospheric moisture
capillary column
compared with packed columns, capillary column offers ____, ____, and ____ but ____
higher resolution
shorter analysis time
greater sensitivity
lower sample capacity
Types of Capillary Column (4)
- Wall Coated Open Tubular Column (WCOT)
- has 0.1 to 5 um thick film of stationary liquid phase on the inner wall of the column - Porous Layer Open Tubular Column (PLOT)
- porous layer (solid particles and liquid coat), such as fused silica, coated onto the column wall - Support Coated Open Tubular Column (SCOT)
- has solid particles coated with a stationary liquid phase - Fused Silica Open Tubular Column (FSOT)
- stationary phase is chemically bonded (still has liquid layer) onto the column wall
In ____ programming, constant oven temperature is used while in ____ programming, it will start at low temperature then gradually ramp to higher temperature
Isothermal - constant oven temperature
Gradient - start at low temperature then gradually ramp to higher temperature
Raising column temperature will (2):
- decreases retention time
- sharpen peaks
a precaution to maintain the life of actual column
Guard Column
used to accumulate and remove non-volatile
substances that would otherwise contaminate (can permanently adhere) the chromatography column and degrade its performance
guard column
Guard column can be ____ since it is replaceable and cheaper
It should be trimmed _____ off, when irregular peaks shapes from a column that had been producing symmetric peaks suddenly appear
cut; 10-20 cm
allows solvent to evaporate prior to entering the chromatography column (separate solvent from solute) to improve peak shapes under certain conditions
retention gap
Desirable properties for the immobilized liquid phase in GLC column (4)
- low volatility
- thermal stability
- chemical inertness
- solvent characteristics fall within a suitable range
Gas chromatographic stationary phases often _____ to _____
bonded and cross-linked
provide longer lasting stationary phase
Detector must be maintained at ____ than the column so analyte will be gaseous
higher temperature
Types of Detectors (8)
- Flame ionization Detector (FID)
- Thermal Conductivity Detector (TCD)
- Electron Capture Detector (ECD)
- Nitrogen Phosphorus Detector (NPD)
- Sulfur Chemiluminescence Detector (SCD)
- Flame Photometric Detector (FPD)
- Photoionization Detector (PID)
- Electrolytic Conductivity Detector (ELCD)
Detector for compounds with C-H bonds
Flame ionization Detector (FID)
Detector used for all compounds except for the carrier gas (Hydrogen, Helium, and Nitrogen)
Thermal Conductivity Detector (TCD)
Detector for halogens, nitrates and conjugated carbonyls (pesticides)
Electron Capture Detector (ECD)
ECD used ____ foil lining the detector cell
Ni-63
Detector for nitrogen and phosphorus containing compounds
Nitrogen Phosphorus Detector (NPD)
Detector for sulfur containing compounds
Sulfur Chemiluminescence Detector (SCD)
Detector for sulfur or phosphorus containing compounds (only one at a time)
Flame Photometric Detector (FPD)
Detector for aromatics and olefins
Photoionization Detector (PID)
Detector for halogens, sulfur or nitrogen containing compounds (only one at a time)
Electrolytic Conductivity Detector (ELCD)
First to be eluted - ____ boiling point
lowest
Qualitative analysis can detect compounds ____
with boiling point up to 400°C
Qualitative analysis ___ detect compounds that are not decomposed at their vaporization temperature but ___ detect highly adsorptive compounds
can detect compounds that are not decomposed at their vaporization temperature
cannot detect highly adsorptive compounds
Qualitative analysis cannot detect ____ compounds and chemically ____ compound
highly reactive and chemically unstable
Qualitative analysis ___ detect compounds that decompose at their vaporization temperature, but always by the same amount solid inert packing
can detect
When analyzed under the same conditions, the same component ______
always elutes at the same time
______ components should be separated, since peaks can overlap with each other and may affect the confirmation of standard
all components
a number, obtained by interpolation (usually logarithmic), relating the adjusted retention time (volume) or the retention time (volume) or the retention factor of the sample component to the adjusted retention times (volumes) of the two standards eluted before and after the peak of the sample component
retention index/ Kovats retention index
used as a second characteristic to reduce false matches but not a sole tool to give specific component
retention index
Retention index usually used ____as standard and applicable to ____
normal alkanes
hydrocarbons
Lowest retention index _____ to elute
first
Given a polar stationary phase, what will be the elution order?
Least polar (non-polar) will elute first
use a separate standard and compare with the unknown
External Standard Calibration
use a separate standard similar in properties with the unknown and you run them together
Internal Standard Method
spike your sample with the same standard
Standard Addition Method
area of each peak is measured and corrected for differences in detector responses to the different eluates.
Peak area method (are normalization method)
similar to external standard but it does not require a separate standard.
Peak integration
introduces the relative sensitivity of the component
Corrected Peak Are Method
Explain traditional liquid chromatography
Good separation and reasonable flow rate but VERY LONG separation
High Pressure Liquid Chromatography has a ___ (4)
good system
small plate heights
narrow peaks
shorter separation times
if lower particle size than HPLC, ____ is used
UPLC (ultra performance LC)
Advantage of HPLC (2)
- can accommodate non-volatile and thermally unstable compounds
- applicable to inorganic ions
HPLC Instrumentation (7)
- Mobile Phase
- Degassing Unit
- Solvent Delivery Pump
- Injector and Sample Vial
- Column and Column Oven
- Detector
- Workstation
Remove dissolved air from mobile phase
degassing unit
Deliver the mobile phase at constant flow
solvent delivery pump
Introduce standard solution or real sample to HPLC column
Injector
Store standard solution or sample solution
Sample Vial
Separate each compound contained in the sample
Column
Keep temperature constant
Column oven
Detect eluted compounds from column
Detector
Signal from detector is processed and chromatogram is displayed
Workstation
____ solvents are required as HPLC mobile phase
Ultra-pure HPLC-grade
Impurities from mobile phase can ____ the column and ___ with detector
degrade
interfere
Solvent should be ___ and ____
filtered and degassed
Dissolved air in solvent may lead to ____ and dissolved O2 would ____
unstable flow rate
absorb UV radiation which would interfere with the spectrophotometric detector
Microfiltration membrane for organic compounds
Teflon
Microfiltration membrane for aqueous solutions
cellulose
Microfiltration membrane for organic and aqueous solution
nylon
Degassing techniques (4)
- Vacuum pumping
- Heating and stirring
- ultrasonicating
- sparging
uses inert gas that is not soluble in the mobile phase to remove dissolved gases
sparging
Modes of solvent delivery (2)
- Isocratic Elution - single solvent
- Gradient Elution (solvent programming) - 2 or more solvents
mode of solvent delivery where solvents are mixed BEFORE ELUTION
isocratic elution
mode of solvent delivery where solvents are mixed DURING ELUTION
gradient elution (solvent programming)
In gradient elution, it uses 2 or more solvent systems that have ____
different polarity
Gradient elution can be used to elute compounds that were _____ on the column
“stuck”
In gradient elution, column has to be ____ after each run because the mobile phase is varied
re-equilibrate
Advantages of gradient elution (4)
- provides equal bandwidths
- fast overall separation
- maximum resolution
- maximum sensitivity
supply the force necessary to move the mobile phase with a constant flow rate and should generate high pressure
pump system
simplest pump available for HPLC that uses motorized syringe to get a constant solvent delivery
Syringe Pump
In using syringe pump, large volume of sample is _____ and has ____ solvent capacity
not required
limited solvent capacity
has small chamber in which the eluent is pumped by the movement of a motor-driven pistol
Single Piston Pump
ensures that eluent will not come back to the chamber
inlet check valve
Disadvantage of single piston pump
not constant flow rate
uses second motor to improve consistency of flow rate use; while one piston is sucking the second motor will release
parallel reciprocating pump
has two motors but has single check valve
series reciprocating pump
provides pulse free output but inexpensive and depends on solvent viscosity and back pressure
gradient pump
different pump is used for each solvent and the eluent composition is determined by the relative flow rate of each pump
binary pump (high pressure mixing)
has one pump but with 4 valves. Eluent composition can be modified depending on which valve is opened
quaternary pump
introduces a small sample (0.-100µL) without depressurization
manual injection
mobile phase bypasses the sample loop and flows directly into the column
load position
mobile phase back flushes the sample from loop into the column
inject position
produce sample into high pressure mobile phase flow without an interruption to the flow
automatic injection (autosampler)
park the sample in a sample loop while the flow is maintained through the column
Sampling Valve
rotates as the sample is introduced into the high pressure system
switching valve
protects the analytical column by removing particulate matter and contamination that might bind to the stationary phase
guard column
column that is made up of tightly packed material; longer than guard column
analytical column
_____ the column can decrease retention time and improve resolution
heating
Property of analytical column that is for the high-pressure resistance (20,000 psi) and expensive
stainless steel
Cheaper alternative for stainless steel analytical column
heavy-wall glass
Property of analytical column that is less pressure (< 600 psi) tolerant but highly resistant to thermal degradation and can easily be cut
PEEK (poly ether ether ketone) Tubing
Property of analytical column that has high speed and minimum solvent consumption. shorter in length and larger in number of plates
Microcolumn
Property of analytical column that separates
components of a mixture and collects isolated components, and non-destructive of the sample
Preparative column
Detector selectivity is ____ important in liquid chromatography than in GC
more
Disadvantages of liquid chromatography
more band broadening and more broad peaks
measures property of mobile phase with or without solute.
Bulk Property (Universal Detector)
Bulk property (universal detector) will____ since it is dependent on the mobile phase
not detect if it is an impurity or solute
measures the difference between refractive index between column eluent and reference stream of pure solvent
refractive index detector
refractive index detector ____ in gradient elution
useless
detection is based on the scattering of a beam of light by particles of compound remaining after evaporation of mobile phase
Evaporative Light Scattering Detector
Evaporative Light Scattering Detector ___ all analyte
Refractive Index Detector ____ to all analyte
Evaporative Light Scattering Detector detects all analyte
Refractive Index Detector responds to all analyte
measures property of mobile phase without the solute and will depend on the property of solute
solute property (selective detector)
most common method that is used to detect sample components which have chromophores that will absorb UV or visible light
UV-Vis Absorbance Detector
very sensitivity detector that is used for sample with fluorescing species
fluorescence detector
detection is based on amperometry, polarography, coulometry which is used to detect electrochemically active species that are easily oxidized/reduced
electrochemical detector
used for ionic compounds that measures electrical resistance which is directly proportional to concentration of ions in the solution
Conductivity Detector
Detector for optically active compounds
Optical Rotation Detector
measures the angle of rotation of polarized light caused by an optical active isomer
polarimetry
Polarimetry is useful for _____ compounds with no absoprtion
chiral
based on difference in refractive index (solute)
Optical Rotatory Dispersion (ORD)
differentiate enantiomers by measuring difference between the absorption of right and left-handed circulatory polarized light due to the existence of a chiral chromophore
Circular Dichroism (CD)
converts analytes to a charged state on the basis of their mass to charge ratio and used when there is no standard
Mass Spectrometry Detector
if refractive index and UV-vis absorbance are used at the same time, _____ can be detected and analyzed
almost ALL components
What to do when column deteriorate?
check if the column exceeded its lifespan
What to do when inappropriate sample solvent or injection volume is applied?
reduce sample volume
The cause of dead volume in the flow lines
irregular flow of the mobile phase due to gas particles in the solvent
Classification of HPLC where separation is based on polarities and the stationary phase is liquid which is immiscible with the liquid mobile phase
Partition Chromatography
liquid is held in place by physical absorption
Liquid-liquid Partition Chromatography
liquid is attached by chemical bonding
Liquid-bonded Phase Chromatography
type of partition HPLC which has polar analyte, polar stationary phase, non-polar mobile phase
Normal Phase Chromatography (NPC)
R groups used to modify the polarity of the stationary phase in NPC (3)
- R=cyano (least polar)
- amino
- diol
In NPC, increasing polarity of mobile phase will ____ while in RPC, increasing polarity of mobile phase will _____
NPC: decrease elution time
RPC: increase elution time
In NPC, ____ polar will be eluted first while in RPC, ____ polar will be eluted first
NPC: least polar
RPC: most polar
Type of partition HPLC which has non-polar analyte, non-polar stationary phase, polar mobile phase
Reverse Phase Chromatography (RPC)
R group of the stationary phase in RPC (3)
All non-polar
1. C8 (n-octyl)
2. C12 (n-dodecyl)
3. C18 (n-octadecyl)
Analyte polarity trend
Hydrocarbon < ethers < esters < ketones < aldehydes < amines < alcohols < salts
Mobile Phase Polarity trend
water > methanol > ethanol > acetonitrile > tetrahydrofuran > propanol > cyclohexane > hexane
Stationary Phase Polarity Trend
Silica > CN > C8 > C18
if %v/v of methanol is increased then its polarity ____ thus retention factor also ____
decreases
decreases (shorter retention time)
separation of relatively polar, water soluble organic compound w/ MW<5000
adsorption chromatography
In adsorption chromatography, stationary phase is a ____ and mobile phase is ____
stationary phase is a polar solid (silica or alumina)
mobile phase is non-polar liquid (single or mixture solvent)
measure of the solvent absorption energy
eluent strength
more polar, _____ eluent strength, the ____ will solutes be eluted from the column
more polar, greater eluent strength, the more rapidly will solutes be eluted from the column
In RPC, eluent strength increases as polarity of solvent _____ while in NPC, increase in eluent strength as solvent becomes ____
decrease
more polar
used to separate molecules of different molecular sized and shapes
Size exclusion chromatography
molecular exclusion
molecular sieve chromatography
size exclusion chromatography for polar water-soluble compounds
gel filtration
size exclusion chromatography for nonpolar compounds
gel permeation
In size exclusion chromatography, ____ molecules will elute first
large
subset of reverse phase chromatography in which ionizable species are separated on reverse-phase columns
ion-pair chromatography
mixture of similar charged ions (polar molecules) separated using ion exchange resin
ion-exchange chromatography
has a separate suppressor column after the analytical column
Suppressor-based/ suppressed ion chromatography
ion exchange chromatography for
separation of negatively charged molecules
anion exchange chromatography
ion exchange chromatography for
separation of positively charged molecules
cation exchange chromatography
In anion exchange chromatography, stationary phase is ____ charged while in cation exchange chromatography, stationary phase is _____ charged.
anion exchange chromatography: positively charged stationary phase
cation exchange chromatography: negatively charged stationary phase
organic polymer with porous structure
ion exhcnage resin
polyvalent ions are ____ held by the stationary phase than singly charge species
much more strongly
If same charge, _____ molecules will elute first
smaller
Ion exchange capacity is dependent on the _____
total number of ion active groups
most specific chromatography with very high selectivity and resolution which is used for separation of biochemical mixtures based on a highly specific interaction between pairs of biological materials
Affinity chromatography
sample is reacted with another reagent to make it compatible with the separation method
sample derivatization
