MedChem Exam 4: Extraction Chromatography

HPLC, GC, Extraction

What percent of drug can you extract in a single extraction if the partition coefficient is 5 and you have 5 ml of patient plasma and 5 ml of ether? Enter the value as a the nearest whole number without the percent sign.

83%

5.7

Rofecoxib

Analytes partition between the mobile phase and the stationary phase.

Diazepam

Biological samples are

complex mixtures that have drug+proteins+sugars+lipids+nucleic acid

Steps of sample analysis

1. Biological samples are collected

2. Purification: Extraction or Chromatography

3. Detection: Spectroscopy or Mass Spectrometry

What do we want to achieve through sample purification step?

remove contaminants that interfere with the identification/quantification of the drug or its metabolites

2 goals during sample purification

1. maximize yield / minimize loss

2. minimize processing time

Describe 2 different sample purification steps

1. extraction or back extraction: isolates drug + other organics

2. chromatography: ideally separates mixture into individual molecules

Name 2 different chromatography methods

1. HPLC ( High Pressure Liquid Chromatography)

2. GC (Gas Chromatography)

During Detection step, what do we detect and how?

Detection of small molecule, typically with quantitation

During Liquid-Liquid extraction, what conditions are remain constant?

temperature and pH

What type are divides in two immiscible solvents: aqueous and organic? and what does it represents?

any neutral, non-dissociating chemical species

ratio of the concentration or the partition coefficient (P=[org]/[aq])

Describes the steps of liquid-liquid extraction

1. aqueous biological sample

2. add immiscible organic phase (not forming a homogenous mixture when added; often octanol or ether)

3. shake to increase surface contact (molecules partition by polarity)

4. isolate target layer

Formula used to calculate the fraction of drug in organic phase

f_org= PV/PV+1

• P: partition coefficient—> given

• V: Volume(org)/ Volume (aq)—> experimentally determined

How do we maximize the yield of liquid liquid extraction? (Hint: 2 different ways)

1. larger volume of organic phase

2. multiple extractions: extract multiple times by adding organic solvent (if target drug is dissolved in this solvent) to the remained aqueous solvent

When does multiple extractions are worth it?

when we quantifying something very small

When do we use back extraction method

ionizable compound that can be manipulated by pH

Describe the steps of back extraction (acid drug)

1. make compound neutral for extraction into an organic solvent —> adding acid makes drug protonated

2. then make the compound ionized to back extract into aqueous—> adding base makes. drug deprotonated

3. e.g. drug pka is 4

   1. pKa>pH: HA

   2. pKa<pH: A-

Describe the steps of back extraction (base drug)

1. make compound neutral for extraction into an organic solvent —> adding base makes drug deprotonated (neutral)

2. then make the compound ionized to back extract into aqueous—> adding acid makes drug protonated (charged)

3. e.g. drug pka is 4

   1. pKa>pH: BH+

   2. pKa<pH: B

What is chromatography?

separation of closely related molecules of complex mixtures based on partition coefficient between a mobile phase with sample in either gas or liquid and stationary phase with similar physiochemical characteristics to sample components to be separated

sample that is mixed as liquid goes to

HPLC

Two different methods of mobile phase forced through a stationary phase

column & plannar

Describe the column chromatography using analyte, mobile phase, stationary phase, migration rate

1. analyte with two or more molecules in the top of the column applied to the stationary phase held in a column by introducing mobile phase

2. Mobile phase flows through stationary phase

3. Migration rate of different molecules in analytes differ depending on the time spent/ affinity in stationary phase vs mobile phase

the faster the molecule comes out (when stationary phase is packed in the column)

has good affinity with mobile phase

what happens if a molecule is only attracted to stationary phase in TLC paper

does not move= no elution

what happens if two molecules are both only attracted to the mobile phase in TLC paper

no separation=coelution

we need to identify a _____ where analytes have partial partitioning to _____

happy medium

both phases

analytes on the column

• diazepem is nonpolar

• nordiazepam is polar due to NH

mobile phase is nonpolar

• interact with diazepem

stationary phase is polar

• interact with nordiazepam

Thus, diazepam elute first as it has a better affinity with mobile phase and then nordiazepam.

Overall goal of chromatogram is to have _______ peaks and eluting in reasonably ______

well separated and short time

What does each of letters in this graph represents?

t₀: void time or dead time —> time for one volume or no volume is going through column; nothing can go faster than this elution time

t_r: retention time—> the time elapsed from the moment a sample is injected into a chromatographic system until a specific compound is detected at the end of the column; change depending on the molecules affinity to phases

w_t: broader base width—> broader with longer retention time if consistent mobile phase or isocratic is used

Why do we have to short run a chromatography under isocratic mobile phase?

samples that elute later have more wider than the samples that elute earlier, resulting poor sensitivity and bad resolution or detection

What are the factors that affects chromatography? (hint: 3)

Efficiency

Retention factor or capacity factor

Selectivity or separation factor

What dose efficiency represents and used?

how well components in analytes are separated and compare columns

how does efficiency is expressed and its formula?

N and N=16 (t_R/W_t)²

N values often (range) with higher number.

High N have a ____ peak at a given retention time than one with lower N

5000-25000

narrower

What are three factors that increase efficiency?

1. longer column

2. smaller stationary phase particle size

3. lower viscosity (fast flow) of mobile phase

How does having

1. longer column

2. smaller stationary phase particle size

3. lower viscosity (fast flow) of mobile phase

contribute to increasing efficiency?

1. longer column = more plates ( more faster partition between two phases)

2. smaller size = fast particle diffusion in stationary phase = shorter tr and narrow width

3. lower viscosity = analytes within mobile phase move faster through stationary phase→ less diffusion over time

What is retention factor or capacity factor (k,k’,k’ A)

• formula

the time a molecule resides with stationary phase relatve to the time it partitions in mobile phase

• formula: k= (t_R-t₀) / t₀

retention factor is increased by (hint: 2 ways)

selecting stationary phase more similar to analyte while less similar to mobile phase

decrease temperature

what is the optimal value of retention time

• having fast or slow retention time can be bad; change depending on the context

>1 at elutes at t₀ and <10 (>10: longer run time, peaks widen)

what is selectivity or separation factor (alpha)

• formula

measure of the time or distance between two peaks

• a= k2 (second peak) / k1 (first peak)

how to increase separation?

changing the mobile phase, stationary phase, running temperature

what is ideal value of separation? what happens is separation=1

a=1 the two peaks co-elute(no separation)

>1.6 is ideal

What does efficiency, retention, selectivity contributes? 

resolution

what is resolution

• formula

ability to separate the peaks considering efficiency, selectivity, and retention

• R_s= route(N)/4 (a-1)/a k/(k+1)

what is the minimum R value for a separation to allow quantitation

R=1

what is desirable for rugged methods

what is baseline separation ensuring the most accurate quantitative result

>_ 1.7 and 1.6

resolution can improve by changing any one of the individual parameters. but the most impactful parameter is 

selectivity

how does selectivity can be changed?

true or false: If the selectivity factor (α) approaches 1 (molecules elute together), then optimizing the capacity factor (k) and the
theoretical plate number (N) doesn’t help a lot to achieve
resolution (see previous slide).

true

two different ways to resolve peaks that had resulted under a constant mobile phase composition (isocratic run) due to mixture being complex:

continuous gradient

step gradient

continuous gradient means 

The mobile phase changes gradually and continuously over time.

step gradient

The mobile phase changes in sudden jumps, not gradually.

the chromatogram of the unknown samples can be determined by the

authentic standards as the same samples under the same conditions are elutes at the same time

how do we quantity a unknown compound

running a known quantity of a standard molecule with the same chromatographic conditions as the unknown.

when comparing an unknown compound quantity using known stand, we compare ___ than ____

compare the peak areas (superior to peak height)

Why is peak area better than peak height for quantitation?

Late-eluting peaks broaden more and become shorter in height, even if the total amount is the same.
➡ Area stays constant, height does not.
➡ So area gives more accurate quantitation.

what are two types of standard used to compare the unknown standard quantity 

external standard and internal standard

what is external standard

• explain the process of getting the concentration of unknown

A known quantity of analyte run separately from the unknown.

You prepare multiple standards → measure peak AREA → plot them → make one calibration curve: peak area vs concentration.

Then you inject the unknown → measure its peak area → plug them in the calibration curve found by standards to calculate the concentration

What do you get when you plug unknown peak area into the external standard calibration equation?

The concentration of the analyte in the unknown sample.

What is an internal standard?

• Steps of internal standard method?

A known amount of a chemically similar internal standard compound that is added to every external standard and unknown samples.
- use linear fit to calculate the concentration in unknown samples

what standard is preferred and why?

internal

Uses the ratio of analyte area / internal standard area, which makes results more accurate as it accounts for anyu sample processing errors.

Two types of chromatography

high pressure liquid chromatography

gas chromatography

high pressure liquid chromatography resolves moleucles in the molecular range of 

100-10,000amu or Da

HPLC type depends on _________ to siloxane polymer particles (hint: 3 different FG)

which functional groups added

• polar 

• nonpolar

• chiral

Normal phase HPLC has a _____ stationary phase and ____ mobile phase

• what are the examples of _____ stationary phase & mobile phase

polar, non-polar

• polar

  • cyano

  • diol

  • amino

  • dimethylamino

• non-polar

  • chloroform

  • n-hexanes

what polarity of molecules elute faster in normal phase HPLC?

nonpolar molecules elute faster

Reverse phase HPLC has a _____ stationary phase and ____ mobile phase

• what are the examples of _____ stationary phase & mobile phase

nonpolar, polar

• nonpolar

  • octyl

  • octyldodecyl

  • phenyl

• polar

  • acetonitrile

  • methanol

  • tetrahydrofuran

what polarity of molecules elute faster in normal phase HPLC?

polar molecules 

the differences in _________________ determine how early/late molecules elute off the column (t_R)

mobile phase polarity

True or False: Enantiomers cannot be separated by reverse or normal HPLC

True

what is enantiomer and diastereomers

enantiomers: non-superimposable mirror images

diastereomer: non-superimposable and non-mirror image

How do we use enantiomer in HPLC

Per-Column Derivatization: Convert Enantiomer to Diastereomers Before HPLC

• React Enantiomeric Mixture with One Enantiomer of a Chiral Derivatizing Agent to Form Diastereomers

If the mixture of S and R used, what happen after adding enantiopure chiral derivatizing compound?

SS and SR which are diastereomer

what happen if racemic mixture A reacts with racemic derivatizing agent B?

• racemic mixture A is S-A and R-A

• racemic mixture B is S-B and R-B

S-A/S-B

R-A/S-B

S-A/R-B

R-A/R-B

R-A/S-B & S-A/R-B —> enantiomers

S-A/S-B & R-A/R-B —> enantiomers

Thus, only two peaks are formed and this is reason why we only need one derivatizing agent 

What is the advantage & disadvantage of pre-column derivatization?

diastereomers resolve on HPLC and increase detection

require sample clean-up and pure derivatizing agent 

what is other solution to separate enantiomer by HPLC?

chiral stationary phase 

but expensive and not often used

How do we optimize normal and reverse HPLC

1. change k—> a —> N order

   1. change k is the easiest to manipulate

   2. how? change mobile phase polarity to be less like analyte

2. gradient: can be continuous or step

What is the mobile and stationary phase of GC?

mobile: inert carrier gas (N2, He, CO2, Ar, H2)

stationary: starts as solid, melted into thin liquid layer coating column walls

What is the physiochemical requirement of sample used in GC

converted to gas phase

what are the physiochemical characteristics of GC? (hint: 2 characteristics)

terminally stable and chemically inert

base is usually siloxane based polymer

What happen to K and t_r of nonpolar solutes when used hydrophobic stationary phase vs hydrophilic stationary phase in GC

larger K (more time in stationary phase) and longer t_r for hydrophobic stationary phase and smaller K and shorter t_r for hydrophilic stationary phase in GC

GC column temperature depends on

In GC, we focus on changing temperature as

analyte BP and degree of separation required

changing the mobile phase characteristics (carrier gas) are not possible

In GC, t_r is dictated by

1. stationary liquid phase

2. column temperature

3. boiling point of the solute/analyte

Explain the GC example in this image

use the ratio to determine the quantity of epitestosterone and testosterone