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Achem 315 > Liquid Chromatography > Flashcards

Liquid Chromatography Flashcards

1
Q

What is special about liquid chromatography

A

The mobile phase is a liquid mixture of solvents

The stationary phase is a silica solid or a bonded phase liquid (all packed there’s no open tubular)

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2
Q

What are the types of liquid chromatography determined by

A

The types of stationary phases focus liek:

The polarity (default)

The analyte molecular size

The ion activity

The specific binding to targets (antibody or chiral)

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3
Q

What type of chromatography is default HPLC

A

It’s partition charmatogeay based on polarity

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4
Q

Why do we need higher pressure in HPLC

A

We need tiny particles because the tinier particles means lower plate heights and fewer multiple paths (the A term in the equation)

This causes narrower peaks and more efficient separation

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5
Q

If the particles were larger in HOLC what would happen

A

The peaks would get broader and slightly slower

QUESTIONS IN NOTRS APP

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6
Q

What is special about the columns for HPLC

A

They’re 2x longer and made of stainless steel to manage the pressure

The diameter of the column determines the flow rate

It has porous silica stationary phase inside that allows the participles to go in and out of (they aren’t trapped)

The efficiency of the column changes as it ages

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7
Q

What is special about the HPLC machine

A

it has a pump that mixes up to 4 solvents to make the eluent which allows us to change the polarity

The column is in an oven that’s about 10 degrees above room temp (east temp to maintain because it needs a constant temp)

In an oven to make high pressure

Made of a guard column and an inline filter

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8
Q

What is the guard column in an HPLC machine

A

A disposable column that collects and filters the permanently binding contaminants that bind to the analyte

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9
Q

What is the in-line filter in a HOLC machine

A

It filters out stuff that’s physically in the solution that could block the column

Comes before the optional guard column

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10
Q

If it’s partition chromatography what does it mean

If adsorption

A

partition: the stationary phase is liquid and the solute partitions/dissolved into it

adsorption: the stationary phase is solid silica and the solute sticks to it

Both a determined by the strength of intermolecular forces

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11
Q

Slide 8

A
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12
Q

What is normal phase liquid chromatography

What is the order of elution

A

The stationary phase is polar

The mobile phase is less polar (or non polar)

The order of elution is that less polar analytes come out first, more polar last because more interaction with polar stationary phase

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13
Q

What is reverse phase liquid chromatography

What is the order of elution

A

The stationary phase is non polar (alkanes ex C18)

The molar phase is more polar (range of polar like 2-propanol (less polar) or water (more polar))

Most polar elutes first

Less polar elutes last

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14
Q

What solvents are normally used in reverse phase liquid chromatography

What about normal phase

A

Water and methanol
More polar mobile phase

Alkanes and tert butyl methyl ether
Less polar mobile phase

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15
Q

If something more polar elites out faster than something less polar what does this mean

A

We’re using more polar mobile phase

Reverse phase

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16
Q

What are isocratic runs

A

When you use one single mobile phase concentrstion/composition throughout the entire hplc run

17
Q

How do we adjust the composition of the mobile phase why

A

We make a solvent gradient

If normal phase, the ending solvent should be more polar to get the most out of the column (same polarity as column)

So we first use a less polar solvent then at the end use a more polar solvent (water)

18
Q

What affects separation in HPLC

A

Temp (effect the kinetics of how thing move off the column and the viscosity of the elunt)

PH (if analyte is protonated/deprotonated it’s interactions with the column change, use buffer to keep constant ph)

Ionic strength (if the solvent supports the analyte breaking into ions, the strength of the analyte affect its interactions of ions with everything)

19
Q

How do we keep the ionic strength constant

A

Add a salt or buffer

20
Q

In liquid chromatography what is pulse prevention

A

As the pump pushes the liquid through, pulses occur where the analyte has pulse in pressure

This causes uneven flow of the analyte

To prevent this, the solvent is pushed in and out to keep a constant pressure with no pulse

21
Q

In liquid chromatography what thisgs actually helps is pulse prevention

A

The valves and pistons in the machine

22
Q

Is the liquid for liquid chromatography injected directly on to the column

What do we use

A

No

An injection loop

23
Q

What is an injection loop

A

It allows for smooth insertion of the liquid and helps deliver a reproducible volume of injection

24
Q

How does the injection valve work in the load position

A

We inject more volume than the loop can hold to fill and rinse it through the sample loop

This solvent is kept in the loop until the inject position

25
Q

How does the injection valve work in the inject position

A

New solvent is sent in and

the pump pushes the stuff in the previously filled sample loop through to the column

The syringe isnt playing a role in it anymore

26
Q

What are the 4 types of detectors in HPLC

A

refractive index

Absorbance

Electrochemical

Mass spectrometry

27
Q

What is the refractive index detector

How does it work

A

Is detects changes in light refraction of the sample

Has the lowest sensitivity but is most common

The solvent (mobile phase) is sent through the two triangular compartments toward a PDA

There is collimated light coming in

When the solute with a different refractive index enters, the light beam shifts and this is detected by the PDA.

28
Q

What is an absorbance detector

A

The path length is small (1cm) in the detector meaning the sensitivity is increased

It becomes smaller by having a crooked path

It’s selective since the response at each different wavelength can be different for the same molecule

Universal because is has a wavelength range that most compounds absorb

29
Q

Can an absorbance detector also be a fluorescence detector?

A

Yes we use the same detector just no light source

30
Q

What a light source the gives uv light

A

Deuterium lamp

31
Q

What is an electrochemical detector

A

It measures the conductivity/redox of the Eluate

Only works for things that are ionized or match a specific value of E

Used electrodes where the sample hits the working electrode

32
Q

What is mass spec detection in HPLC

A

it’s the most specific and sensitive detector

Uses electro spray ionization

Achem 315 (13 decks)

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