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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
**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
26
**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_**
27
**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
28
**UPLC** Ultra Performance Liquid Chromatography
**15k Psi, ~1.7um particle size** **Significant increases in:** **RESOLUTION / SPEED / SENSITIVITY** vs HPLC
29
**As column LENGTH INCREASES** **Seperation resolution does what?**
**_INCREASES_** ## Footnote because there is **MORE TIME** & **MORE OPPORTUNITIES** for the MP to interact w/ the SP
30
***As column particle SIZE decreases*** **what happens to Seperation resolution?**
**_INCREASES_** ## Footnote MORE **_SURFACE AREA**_ & MORE _**PARTICLES_** give more **OPPORTUNITIES TO INTERACT** w/ the SP
31
**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**
32
**Prep**erative **Column**
Allow for **HPLC** in **different major scales** Flow rates **\>10mL/min** Sample INJ size **\>5mg**
33
***Semi-*Prep**erative **Column**
Allow for **HPLC** in **different major scales** intermediate Flow rates ~**1-3mL/min** Sample INJ size **~0.5-5mg**
34
**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**
35
**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
36
**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*
37
**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**_
38
**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 * Also used for: * testing for **biological activity** * **MoA testing**
39
**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
40
**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_***
41
**What type of chromatography is best for** **PROTEIN SEPERATION?**
**_GPC_** **_Size Exclusion Chromatography_**
42
**What type of chromatography/seperation is best for** **CLEANING UP samples?** (for HPLC)
**SOLID PHASE EXTRACTION** **SPE**
43
**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_**
44
**What type of chromatography/seperation technique is best for** **isolating a SPECIFIC SUBSTANCE?**
**AFFINITY CHROMATOGRAPHY** best to isolate specific substance from complicated matrices

BioMedChem M2 (8 decks)

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