18/19 - Chromatography Flashcards
Different Types of CHROMATOGRAPHY
TLC (Thin layer)
Column Chromatography
HPLC (High Performance Liquid)
GC (Gas Chromatography)
Gel-pemeation / Size Exclusion Chromatography
Gas Chromatography
seperate based off of affinity to:
MP = VOLITILATY & SP = ADSORPTION
- Mobile Phase = GAS
- limited to analytes stable in gas phase
- SP = Adsorbent material
- Passed through by pressure (pump)
- Compounds may be chemically altered/derivatized
- to increase their Volatility / Thermal Stability
-
Derivitation can entail:
- methylation of COOH -> methyl esters
- -OH -> ethers
Gas Chromatography (GC) is able to anaylze what?
VOLATILE GASSES
(if you can SMELL it)
Prefer
Small MW
Non-Polar
Liquid Chromatography (LC)
seperate based off their affinity to the:
MP = SOLUBILITY & SP = ADSORPTION
- MP = Dissolved Liquid + Analyte
- Passed over SP = adsorbent material
- Column
- open tubular or pre-packed
- most commonly used to isolate/seperate/analyze molecules
LC is able to analyze what?
wider range of molecules vs GC, since Most pharmaceutical compounds, are NOT volatile
Prefer:
med- Large MW
large range of Polarity
Drugs discovered from NATURE
TAXOL = bark
Ziconotide = ocean, snail
Yondelis = sea tunicate
Morphine = poppy seeds
Quinine = bark
Lovastatin = bacteria
General Uses of Chromatography
Mix of Unknowns -> ID of unknown via comparison to reference
Mix of Compounds -> Single Compound
— for spectroscopy-> structure
3 Common Mechanisms of Seperation:
Partition, analyte–> homogenous solution in each phase
Adsorption,interaction at a surface or fixed sites of SP
Exclusion, ability of a porous solid stationary phase to discriminate analytes by SIZE
What is a Column?
Hardware that hosts the STATIONARY PHASE
Glass** for **Column Chromatography
Metal casing for LC / GC
(to handle the high pressure / performance needs)
Stationary Phase = SP
-
Organic functional groups fused to particles (usually silica)
- interact with analytes passing through the column via:
- H-Bonding
- VDW
- Pi-Pi stacking
- interact with analytes passing through the column via:
- SP broken down into:
- Normal Phase (POLAR)
- Reversed Phase (non-polar)
NP Chromatography
Normal Phase = SP is POLAR
- SP = HydroPHILIC
- made of of relatively POLAR groups
- -OH / -NH2 / -CN
- made of of relatively POLAR groups
- MP = relatively hydrophobic
RP Chromatography
Reversed Phase** = SP is **Non-Polar
- SP = relatively Non-Polar Groups
- C8 / C18 / Phenyl / Phenyl Hexyl
- support hydrophobic interactions
- LIKE LIKE
- C8 / C18 / Phenyl / Phenyl Hexyl
- MP = relatively hydroPHILIC
Plate Theory
Band Broadening can occur & we want to MINIMIZE it
- Column Efficiency:
- Whole passing through column, analyte passes from a:
-
spreads from narrow band to broader band
- = BAND BROADENING
-
spreads from narrow band to broader band
- Whole passing through column, analyte passes from a:
- We want to MINIMIZE this:
-
if band broadening occurs SLOWLY
- different rates of migration -> better SEPERATION!
-
if band broadening occurs SLOWLY
Mobile Phase
MP
- MP will pass through column carrying the analyte(s) of interest:
-
interact w/ both MP & SP to different degrees
- Extent of Interaction : Time Spent on column
-
interact w/ both MP & SP to different degrees
- Stronger MP Solvent
- the more it RESEMBLES POLARITY of SP
Strength of MP?
-
Stronger MP Solvent:
-
one that MOST resembles the POLARITY of the SP
- so it may better compete for Analytes w/ SP
-
one that MOST resembles the POLARITY of the SP
- Stronger Solvent leads to
- = GREATER SEPERATION
Order of increasing Solvent Strength (MP)
Hydrophobic = non-polar Solvents
to be used with NP = polar stationary phase
ISOPROPANOL
Ethyl Acetate
CHCl3 (chlorophorm)
DCM (dichloromethane)
Hexanes
Order of INCREASING Solvent Strength
HydroPHILIC = POLAR Solvents
to be used with RP = non-polar stationary phase
METHANOL (CH3OH, MeOH)
Acetonitrile (CH3CN)
water
Isocratic Flow
type of LC Flow, same MP throughout
- MP = ONE SOLVENT COMPOSITION,
- also the same concentration
-
for the ENTIRE duration of seperation
- used in TLC / Column / HPLC
-
for the ENTIRE duration of seperation
- also the same concentration
Step Gradient Flow
type of LC Flow,
same MP compound, different Concentration
SERIES OF ISOCRATIC STEPS
multiple steps of an Isocratic flow, ex:
1st run = 60% MeOH (methanol)
2nd = 70% MeOH
3rd = 80% MeOH…..
Gradient Flow
type of LC flow, used in HPLC / GC / flash systems
Continuously Changing Solvent System
typically 2 different solvents, gradual change
10% MeOH –> 100% MeOH
over 25 minutes
Open Column = Gravity Column
Chromatography
MP / SP?
type of Column Chromatography
Simplest form, no need for pumps
SP = powder form, pack your own glass column
MP = Solvents carry analytes via GRAVITY
Flash Column
Chromatography
MP / SP?
Identical as Open/Gravity column, EXCEPT:
column is PRESSURIZED
via pump/house air, through TOP
, allows for FASTER analyte seperation
SP = self packed powder glass column
Vacuum Liquid Column
Chromatography
MP / SP?
- nearly ID as flash column, EXCEPT*
- instead of pressure @ TOP,*
VACUUM is attached
BELOW the SP
SP = powder pack
Solid Phase Extraction
SPE
same principles as Column Chromatography, however
SP= columns typically come PRE-PACKED
can be used as a standard seperation of mixtures or….
Clean-Up in preparation for HPLC
removes any UNWANTED materials that might covalently bond
How do we INCREASE chromatographic RESOLUTION?
for packing material = SP
SMALLER PARTICLE SIZE of <10um
lead to BETTER seperation between peaks
- but smaller particles give greater resistance to flow*, since it is more TIGHTLY packed
- => Too Slow*
HIGHER PRESSURES ARE NEEDED –> HPLC
TLC
Thin Layer Chromatography
SP / MP?
common form of PLANAR chromatography, replaced with HPLC
SP = glass/metal/plastic sheet thinly coated with silica gel
MP = developing chamber with solvent,
analyte is spotted on and travels up via
CAPILLARY ACTION
HPLC
High Pressure Liquid Chromatography
Uses a Moderate-High Pressure to push solvent through column
allows for GREAT INCREASES in chromatographic resolution, due to very small particle sizes
Can seperate / ID / quantify analytes such as:
pharmaceuticals / food / cosmetics
chemicals / forensics / naturals
UPLC
Ultra Performance Liquid Chromatography
15k Psi, ~1.7um particle size
Significant increases in:
RESOLUTION / SPEED / SENSITIVITY
vs HPLC
As column LENGTH INCREASES
Seperation resolution does what?
INCREASES
because there is MORE TIME & MORE OPPORTUNITIES for the MP to interact w/ the SP
As column particle SIZE decreases
what happens to Seperation resolution?
INCREASES
MORE SURFACE AREA** & MORE **PARTICLES
give more OPPORTUNITIES TO INTERACT w/ the SP
ID EACH PART OF THIS HPLC
+ degasser = degasses solvents
+ autosampler = automated sampling for concecutive HPLC runs
A- Injection Port
B- Mobile Phase
C- Controller
D- Pumps
E- Column
F- UV detector
Preperative Column
Allow for HPLC in different major scales
Flow rates >10mL/min
Sample INJ size >5mg
Semi-Preperative Column
Allow for HPLC in different major scales
intermediate
Flow rates ~1-3mL/min
Sample INJ size ~0.5-5mg
Analytical Column
Allow for HPLC in different major scales
last / smallest / slowest
Flow rates ~0.2-1 mL/min
Sample INJ size 0.01-0.5 mg
Common Types of Stationary Phases
C-18
long CARBON CHAIN
Silical Gel
many -OH groups
Phenyl SP
many PI-PI interactions
Phenyl Hexyl
Many carbons between the phenyl group
What is a Chromatogram?
a READOUT from a Detector
visual rep. of anylytes that were seperated in the HPLC system
X = Time
Y= typically ABSorbance
Individual Peaks = Analyte/Compound that elute from column & pass thru detector
HIGHEST peak SOMETIMES correlates with quantity of compound present on the molecule
Uses for the CHROMATOGRAM
Identifying Unknown compounds
SPOT FAKE DRUGS
compare tested HPLC Chromatogram vs a STANDARD
- HPLC is often used to identify compounds from a mixture of unknowns.
- by comparing the tr (retention time) with an reference standard.
- MUST USE THE SAME CHROMATOGRAPHIC SYSTEM & SAME EXPERIMENTAL PROTOCOL
- by comparing the tr (retention time) with an reference standard.
Uses for the Chromatogram
Isolating pure compounds
- HPLC is also used to isolate pure compounds
- Individual peaks can be COLLECTED as they elute from the detector
- as long as NON-DESTRUCTIVE detection is used
- semi-prep / prep HPLC
- as long as NON-DESTRUCTIVE detection is used
- Also used for:
- testing for biological activity
- MoA testing
GPC / Size Exclusion Chromatography
Gel Permeation
useful for seperating compounds that are fairly
VARIENT IN SIZE
- In analyte mixture:
- Smaller Compounds enter pores in the SP beads
-
Larger Compounds are *excluded* from the pores
- carried through column faster by the MP
- GPC columns:
- variety of pore sizes, relative to MW cutoffs
USES for
GPC / Size Exclusion Chromatography
Gel Permeation
seperating based on how big or how small compounds are
PROTEIN SEPERATION
since there is an absence of analyte-SP interactions
prevents:
denaturing / change in CONFORMATION
loss of _biological activity_
What type of chromatography is best for
PROTEIN SEPERATION?
GPC
Size Exclusion Chromatography
What type of chromatography/seperation is best for
CLEANING UP samples? (for HPLC)
SOLID PHASE EXTRACTION
SPE
Affinity Chromatography
one of the MOST SPECIFIC seperation techniques
uses a custom made SP designed to bind/retain
ONE PARTICULAR ANALYTES
ex. selective isolation of biotin
What type of chromatography/seperation technique is best for
isolating a SPECIFIC SUBSTANCE?
AFFINITY CHROMATOGRAPHY
best to isolate specific substance from complicated matrices
