HPLC

HPLC (aka "partition chromatography") is the most widely used analytical separation technique because most small molecules fall into the range of molecular sizes that HPLC can resolve. What is this range?

100-10000 amu/Da

Explain a gradient

A gradient is where you switch from one mobile phase ratio to another over time (continuous gradient or step).

When derivatization is used to improve resolution of a mixture of analyte, it must be performed: A. precolumn only B. postcolumn only C. precolumn or postcolumn D. derivatization is used to improve detection only

A. precolumn only

A molecule has two chiral centers. Compound A is the RS isomer, while compound B is the RR isomer. Which of the following is true? A. The two compounds are enantiomers B. The two compounds are diastereomers C. They are optically inactive D. Can not tell the type of stereoisomers

B. The two compounds are diastereomers

For most drugs, which general method of liquid chromatography is employed? A. adsorption chromatography B. partition chromatography C. ion-exchange chromatography D. size exclusion chromatography

B. partition chromatography

Why does the derivatizing agent have to be enantiopure?

Because less peaks will be detected due to enantiomer pairs if it were a racemic mixture. This means separate compounds wouldnt be detected.

How does changing k'A affect α?

Because the larger the k'A, or optimal migration rate of solute, the larger the selectivity factor (less of resolution).

If you have two closely related drugs that show poor separation on reverse HPLC, which of the following would be the best option for improving resolution? A. Use ion pairing chromatography B. Derivatize with ninhydrin C. Increase the polarity of the mobile phase D. Decrease the polarity of the mobile phase E. None of the above

C. Increase the polarity of the mobile phase

Which common HPLC mobile phases are polar?

CH3CN, CH3OH, and THF (tetrahydrofurane)

Which common HPLC mobile phases are nonpolar?

CHCl3 and C6H6

Describe how a chiral stationary phase can be used to separate chiral molecule.

Chiral molecules would be more attracted to the chiral stationary phase therefore it would allow for varied elution. The stationary phase has a 3-D structure that will interact differently with different stereoisomers.

Common HPLC polar stationary phases

Cyanos (CN), diols, and amino groups

In reverse phase HPLC, which is true? A. The stationary phase is usually polar in nature. B. We can only use polar analytes. C. The analyte that elutes first is the least polar of the analytes in the sample. D. The analyte that elutes first is the most polar of the analytes in the sample.

D. The analyte that elutes first is the most polar of the analytes in the sample.

What are the advantages to pre-column derivatization to separate enantiomers method?

Diastereomers resolve on HPLC and it improves detection

how can you use pre-column derivatization to separate enantiomers??

If our compound of interest is not a diastereomer, then we can make it a diastereomer prior to HPLC by using this method. In the class example, we have ibuprofen (R and S enantiomers) and can react it with some other chiral, enantiopure derivatizing agent being X. If X were S, then we would make RS and SS which are diastereomers of each other and can be separated.

Explain derivatization

It is a 100% chemical modification of the analyte to alter its polarity as desired by incorporating a chromophore via dye group to make it colored. This can enhance the detector response to certain components in a mixture. In the case of adding a chromophore, you can now detect analyre via absorbance.

What is the best variable to manipulate when optimizing HPLC separation?

K'A (capacity factor)

What types of molecules are best suited for ion-pair reverse phase HPLC?

Molecules that are highly charged.

Explain how the relative polarity of the mobile phase affects elution time in normal phase

Nonpolar molecules elute earlier than polar molecules. Relative increases in mobile phase polarity decreases the retention time.

What mathematical equation is used to change the mobile phase composition

PAB =øAPA +øBPB

In normal phase HPLC, is the stationary phase polar or nonpolar?

Polar

Explain how the relative polarity of the mobile phase affects elution time in reverse phase

Polar molecules will elute earlier than the nonpolar molecules. Relative decreases in mobile phase polarity decreases retention time.

Is pre- or post-HPLC derivatization preferred? Why?

Post-HPLC derivatization is preferred. This is because it's more work to do pre-HPLC due to having to re-optimize experimental HPLC conditions due to chemical alteration, it's chemically different than what you optimized for

upercritical fluid chromatography (SFC) is a "hybrid" of gas chromatography and liquid chromatography because the mobile phase is considered liquid. Describe the mobile and stationary phase in SFC and explain why temperature plays an important role.

SFC uses normal phase chromatography in which the mobile phase is made up of CO2 (nonpolar - similar polarity to heptane) and SiO2 (polar) stationary phase as well as a cosolvent such as methanol to modify polarity.

How can stereochemistry affect/determine the therapeutic and/or toxic effects of drugs?

Stereoisomers are two different molecules and therefore can exhibit 2 different effects within the body. For example, the R stereoisomer of thalidomide has been used previously to alleviate nausea in pregnant women while the S stereoisomer causes birth defects in children. Another example would be that the R stereoisomer of a compound is Dextromethorphan which is an OTC antitussive while the R stereoisomer is Levomethorphan which is a controlled narcotic

what does the equation PAB =øAPA +øBPB tell you?

This allows you to increase the selectivity factor by changing the mobile phase composition while keeping migration rate of solutes constant.

What situations is derivatization used for?

This is only really used if you can't change the stationary or mobile phase. It's seen as a last resort method to optimize separation.

Which types of compounds are best suited for SFC?

Used for separation of chiral molecules OR achiral purifications.

What kinds of situations is a gradient used for

Useful If you have good resolution where one solute elutes early and then a long period of time where nothing elutes and then 2nd and 3rd compounds come off. This would be more beneficial if you had your column set to the same composition for the first compound and then gradually change it for the 'dead' period to get the latter 2 compounds to get off earlier.

How can you change mobile phase composition?

You can do this by increasing the mobile phase polarity so polar solutes elute much quicker than non-polar. (ex. If reverse phase, make more polar. If normal phase, make more nonpolar)

Ion pair reverse phase HPLC

allows one to use reverse phase HPLC with highly charged molecules, in which the nonpolar column retains an ionic compound and the mobile phase contains a reagent with both an ionic and a hydrophobic end. The reagent gets absorbed by the stationary phase, then interacts with the charged molecule to afford some retention.

How can we optimize an HPLC run?

change mobile phase, gradient elution, derivatize analyte

Why can't HPLC be used to separate enantiomers, but they can be used to separate diastereomers? (hint: think about whether groups are on the same side relative to each other)

enantiomers have similar properties and polarity so it won't show a difference between them in HPLC hence why if you used a non enantiopure derivatizing agent with a compound that exhibits both R and S there would only be 2 peaks instead of 4 separate peaks, the enantiomer pairs would look the same/show the same results.

In HPLC, a siloxane polymer with long hydrocarbon side chains is considered a ________ stationary phase and will lead to _________ retention times for nonpolar analytes. A. hydrophobic or hydrophilic B. shorter or longer

hydrophobic, longer

how would you decide between normal and reverse phase HPLC?

if we have 2 compounds that are relatively nonpolar, then (reverse phase) nonpolar column is the better option. if we have 2 compounds that are intrinsically polar, then normal phase is the better choice

Diastereomers

molecules that contain multiple chiral centers are non-superimposable, non-mirror images.

Enantiomers

molecules that contain one more more chiral centers that are mirror images of eachother.

In reverse phase HPLC, is the stationary phase polar or nonpolar?

non polar

Common HPLC nonpolar stationary phases

octyl (C8H17), C18H37, and C6H5

What are the disadvantages to pre-column derivatization to separate enantiomers method?

requires sample cleanup, requires pure derivatizing agent, and can distort the "true" ratio of enantiomers

How does ion-pair reverse phase HPLC work?

the nonpolar column retains an ionic compound in this method (doesn't normally) and the mobile phase contains a reagent with both an ionic and a hydrophobic end. The reagent gets absorbed by the stationary phase, then interacts with the charged molecule to afford some retention. If the drug is positive, you would want a negative reagent. If the drug is negative, you would want a positive reagent.