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BioMedChem M2 > 18/19 - Chromatography > Flashcards

18/19 - Chromatography Flashcards

1
Q

Different Types of CHROMATOGRAPHY

A

TLC (Thin layer)

Column Chromatography

HPLC (High Performance Liquid)

GC (Gas Chromatography)

Gel-pemeation / Size Exclusion Chromatography

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2
Q

Gas Chromatography

A

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
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3
Q

Gas Chromatography (GC) is able to anaylze what?

A

VOLATILE GASSES

(if you can SMELL it)

Prefer

Small MW

Non-Polar

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4
Q

Liquid Chromatography (LC)

A

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
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5
Q

LC is able to analyze what?

A

wider range of molecules vs GC, since Most pharmaceutical compounds, are NOT volatile

Prefer:

med- Large MW

large range of Polarity

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6
Q

Drugs discovered from NATURE

A

TAXOL = bark

Ziconotide = ocean, snail

Yondelis = sea tunicate

Morphine = poppy seeds

Quinine = bark

Lovastatin = bacteria

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7
Q

General Uses of Chromatography

A

Mix of Unknowns -> ID of unknown via comparison to reference

Mix of Compounds -> Single Compound

— for spectroscopy-> structure

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8
Q

3 Common Mechanisms of Seperation:

A

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

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9
Q

What is a Column?

A

Hardware that hosts the STATIONARY PHASE

Glass** for **Column Chromatography

Metal casing for LC / GC

(to handle the high pressure / performance needs)

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10
Q

Stationary Phase = SP

A
  • Organic functional groups fused to particles (usually silica)
    • interact with analytes passing through the column via:
      • H-Bonding
      • VDW
      • Pi-Pi stacking
  • SP broken down into:
    • Normal Phase (POLAR)
    • Reversed Phase (non-polar)
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11
Q

NP Chromatography

A

Normal Phase = SP is POLAR

  • SP = HydroPHILIC
    • made of of relatively POLAR groups
      • -OH / -NH2 / -CN
  • MP = relatively hydrophobic
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12
Q

RP Chromatography

A

Reversed Phase** = SP is **Non-Polar

  • SP = relatively Non-Polar Groups
    • C8 / C18 / Phenyl / Phenyl Hexyl
      • support hydrophobic interactions
      • LIKE LIKE
  • MP = relatively hydroPHILIC
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13
Q

Plate Theory

A

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
  • We want to MINIMIZE this:
    • ​if band broadening occurs SLOWLY
      • different rates of migration -> better SEPERATION!
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14
Q

Mobile Phase

MP

A
  • 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​​
  • Stronger MP Solvent
    • the more it RESEMBLES POLARITY of SP
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15
Q

Strength of MP?

A
  • Stronger MP Solvent:
    • ​one that MOST resembles the POLARITY of the SP
      • so it may better compete for Analytes w/ SP
  • Stronger Solvent leads to
    • = GREATER SEPERATION
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16
Q

Order of increasing Solvent Strength (MP)

Hydrophobic = non-polar Solvents

A

to be used with NP = polar stationary phase

ISOPROPANOL

Ethyl Acetate

CHCl3 (chlorophorm)

DCM (dichloromethane)

Hexanes

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17
Q

Order of INCREASING Solvent Strength

HydroPHILIC = POLAR Solvents

A

to be used with RP = non-polar stationary phase

METHANOL (CH3OH, MeOH)

Acetonitrile (CH3CN)

water

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18
Q

Isocratic Flow

A

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
19
Q

Step Gradient Flow

A

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…..

20
Q

Gradient Flow

A

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

21
Q

Open Column = Gravity Column

Chromatography

MP / SP?

A

type of Column Chromatography

Simplest form, no need for pumps

SP = powder form, pack your own glass column

MP = Solvents carry analytes via GRAVITY

22
Q

Flash Column

Chromatography

MP / SP?

A

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

23
Q

Vacuum Liquid Column

Chromatography

MP / SP?

A
  • nearly ID as flash column, EXCEPT*
  • instead of pressure @ TOP,*

VACUUM is attached

BELOW the SP

SP = powder pack

24
Q

Solid Phase Extraction

SPE

A

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

25
Q

How do we INCREASE chromatographic RESOLUTION?

A

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

26
Q

TLC

Thin Layer Chromatography

SP / MP?

A

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

27
Q

HPLC

High Pressure Liquid Chromatography

A

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

28
Q

UPLC

Ultra Performance Liquid Chromatography

A

15k Psi, ~1.7um particle size

Significant increases in:

RESOLUTION / SPEED / SENSITIVITY

vs HPLC

29
Q

As column LENGTH INCREASES

Seperation resolution does what?

A

INCREASES

because there is MORE TIME & MORE OPPORTUNITIES for the MP to interact w/ the SP

30
Q

As column particle SIZE decreases

what happens to Seperation resolution?

A

INCREASES

MORE SURFACE AREA** & MORE **PARTICLES

give more OPPORTUNITIES TO INTERACT w/ the SP

31
Q

ID EACH PART OF THIS HPLC

A

+ 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

32
Q

Preperative Column

A

Allow for HPLC in different major scales

Flow rates >10mL/min

Sample INJ size >5mg

33
Q

Semi-Preperative Column

A

Allow for HPLC in different major scales

intermediate

Flow rates ~1-3mL/min

Sample INJ size ~0.5-5mg

34
Q

Analytical Column

A

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

35
Q

Common Types of Stationary Phases

A

C-18

long CARBON CHAIN

Silical Gel

many -OH groups

Phenyl SP

many PI-PI interactions

Phenyl Hexyl

Many carbons between the phenyl group

36
Q

What is a Chromatogram?

A

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

37
Q

Uses for the CHROMATOGRAM

Identifying Unknown compounds

A

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
38
Q

Uses for the Chromatogram

Isolating pure compounds

A
  • 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
  • Also used for:
    • testing for biological activity
    • MoA testing
39
Q

GPC / Size Exclusion Chromatography

Gel Permeation

A

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
40
Q

USES for

GPC / Size Exclusion Chromatography

Gel Permeation

A

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_

41
Q

What type of chromatography is best for

PROTEIN SEPERATION?

A

GPC

Size Exclusion Chromatography

42
Q

What type of chromatography/seperation is best for

CLEANING UP samples? (for HPLC)

A

SOLID PHASE EXTRACTION

SPE

43
Q

Affinity Chromatography

A

one of the MOST SPECIFIC seperation techniques

uses a custom made SP designed to bind/retain

ONE PARTICULAR ANALYTES

ex. selective isolation of biotin

44
Q

What type of chromatography/seperation technique is best for

isolating a SPECIFIC SUBSTANCE?

A

AFFINITY CHROMATOGRAPHY

best to isolate specific substance from complicated matrices

BioMedChem M2 (8 decks)

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