chromotagraphy quiz

Question 1

what is chromotagraphy

Answer 1

It is a techinque used to seperate a mix of products. It works on the principle that different products in the mixture will distrubte enequally between two immiscible phases (mobile and stationary) since they have different degress of atttraction to the stationary phase.

Question 2

which product will travel more slowly down the stationary phase and which one faster?

Answer 2

The one that will travel more slowley is the one most attracted to the stationary phase as it will bond to it for longer and stronger so it will travel more slowly. The product that has weak attractions to the stationary phase will end up traveling faster.

Question 3

define mobile phase and stationary phase

Answer 3

mobile: the solvent moving through the column

stationary: the substance that stays fixed inside the column

Question 4

define eluent vs eluate

Answer 4

eluent: is the mobile phase entering the column

eluate: is the mobile phase exiting the column, what is collected in the flasks

Question 5

define elution

Answer 5

the process of washing out a compound through a column using a suitable solvent

Question 6

define analyte

Answer 6

the molecule we want to seperate from mixture

Question 7

what do both retention time and volume refer to and define each one sepertely

Answer 7

they both refer to the duration that an analyte spends on the chromotagraphy column

1. retention time: is the time it takes from injection of analyte until release/ collection
2. retention volume: the amount of mobile phase pumped onto the column between analyte injection and analyte detection

Question 8

define column chromotagraphy and its uses

Answer 8

It is used on the both large and small scale to seperate and purify material. it seperates based on the differential partitioning between mobile and stationary phase.

Question 9

is silica gel polar?

Answer 9

yes its made up of a lot of OH

Question 10

whats the difference between isocratic and gradient elution

Answer 10

isocratic elution uses one solvent at the same concentration so the mobile phase composition is kept constant, so the analyte componentes will eulute seperatley according to their affinity to that single solvent

gradient elution uses more than one solvent, so the composition of the mobile phase changes during the chromotgraphic run. This is more commonly used when the mixture is known to have a wide range of retention factors to be seperated.

Question 11

whats an example of a polar and non polar mobile phase

Answer 11

nonpolar: hexane

polar: acetonitrile

Question 12

what types of chromotrgaphy are based on polarity?

Answer 12

1. hydrophobic interction chromotgraphy→ hydrophobic is non polar
2. normal or rerverse phase chromotgraphy→ polarity, (normal= polar, reverse=non polar)

Question 13

what are the other three types of chromotrgaphy not based on polarity?

Answer 13

1. ion exchnage chromotrgaphy (net charge)
2. affinitiy chromotrgaphy (based on specific binding)
3. size exlusion: based on molecular size

Question 14

what is the role of salt in hydrophobic interaction chromotragphy?

Answer 14

1. salt allows for the interaction between hydrophobic plate and hydrophobic groups
2. reducing the salt concentration allows for seperation/filtering out based on hydrophobicity

Question 15

describe the principle of hydrophobic interaction chromotraphy and hows its done

Answer 15

It seperates molecules based on their hydrophobicity. Its based on the fact thet hydrophobic molecules will bind to hydrophobic stationary phase in the presence of high [] of salt, since it enhances hydrophobic interactions.

Question 16

how does salt actually work in hydrophobic chromotrgaphy

Answer 16

So at first the water in the mobile phase will form a dense layer around the analyte molecules and the hydrophobic groups on the column basically shielding them from interaction. But by adding salt you break the polar hydrogen bond network that formed, which will expose the hydrophobic groups to the stationary phase which allows for the biniding. You wold decrease the [] to allow for the moleucles to filter out, the fiirst to elute are the least hydrophobic and the rest will follow based on their level of hydrophobicity.

Question 17

what is hydrophobic interaction chromatraphy used for?

Answer 17

for the seperation of large biomolecules like protien, because with this method you dont need organic solvents which denature protiens instead you can use aqueous medium

Question 18

whats the difference between normal and reverse phased chromotraphy and how do their stationary phases differ

Answer 18

• normal phase: has a highly polar stationary phase like silica gel which means polar molecules will be retained in the column and non polar molecules will elute first
• reversed phase: has a highly non polar stationary phase like C18 which retain non polar compounds and the polar molecules will elute first

Question 19

what is the basis of ion exchange theory and what are the two types?

Answer 19

Opposite charges attact each other, and similar charges repel each other, so seperation is done based on their ionic interactions with one another.

1. Anion-exchnage chromotragph: want to purifiy -ve so you pass it through a positively charged resin
2. cation-exchange chromotraphy: you want to purify +ve charged analyte so you pass it through a negativelu charged resin

Question 20

what are the are the four types of ion-exhcnage resisn and how do they differ in their functional group?

Answer 20

1. strong cation: strong acid, normally has S group
2. strong anion: strong base, normally features a quatenery amino group (Q)
3. weak cation: weakly aciid, normally has a carboxylic acid group
4. weak anion: weakly basic, normally has primary, secondary and or tiertary amino groups

Question 21

if the target molecule are stable at a pH value lower or higher than pI then what are the corresponding exchangers needed

Answer 21

→ lower: cation exchnager prefereed, because below pI is acidic (H+)

→ higher: anion exchnager prefered, because above pI is basic (OH-)

Question 22

describe affinity chromotrapghy

Answer 22

It is a lock and key mechanism where the analyte is the only key with the complemntary shape to fit in the “lock” on the stationary phase. this allows you to flish out unwanted species and when your ready you just break that lock and key interaction to collect analyte.

Question 23

Describe size exlusion chromatraphy

Answer 23

This is a process where molecules are seperated based on szie through a hydrophilic polymer. SO large protiens will come out first as they cannot fit in the pores and small protiens will come out last, so they get stuck in the pores.

Question 24

what is GC suited for the analysis of and what is an example?

Answer 24

For volatile substances which are thermally stable such as fatty acids, triglycerids etc

Question 25

what are the three general steps of GC with lipids

Answer 25

1. extracting the lipids or target 2. Derivation: basically this step makes the product volatile. So triglcyerides and phospholipids are saponified and the free fatty acids will be esterified forming FAMES 3. analyzation using capillary gas chromatraphy

Question 26

What is the principle of GC/actual steps of proccess

Answer 26

A sample will be injected into gas chromogtraphy and a mobile phase which is a carrier gas will move throigh the column containing the stationary phase which is under controlled temperature gradient. As it moves through the components will be distrubted between the two phases. The seperation is based on differences in properties and structure, resulting in a differential migration of solutes leading to seperation..

Question 27

what does the machinery of GC composed of?

Answer 27

1. flowing mobile phase 2. injection port 3. seperation column 4. oven 5. dector

Question 28

what is the carrier gas normally in GC and why can it not be oxygen or air

Answer 28

It is normally helium, nitrogen, hydrogen because it must be chemically inert, free and dry of O2 so therefore air cannot be one or oxygen, and this is because if not it can react with the sample

Question 29

what is the sample injection system and what are 2 types?

Answer 29

It is a heated block where the sample will be injected. It is maintained at a temperature higher than the boiling point of the least volatile part of the sample. Two types include: 1. autosampler: sample is automatically injected into inlets 2. split/spiltess injector: sample is introduced into injector and the carrier gas either sweeps the entire sample so spiltless mode or a portion (split) of the sample into the column

Question 30

what is spilt mode preffered for and when is spitless prefered

Answer 30

split→ high analyte concentrations, as a part of the sample will be exhausted through vent splitless→ best for trace anlaylis with low amounts of analytes

Question 31

where are columns stored and why

Answer 31

In thermostated oven because controlling them temp is essential for good speration

Question 32

what are common types of dectors and explain FID

Answer 32

Types include thermal conductivity decteor (TCD), nitrogen phosphorus dectors (NDP), electron capture detector (ECD) and finally FID, flame ionization detector. FID uses a flame to ionize organic compounds containing carbon. So following seperation each analyte will pass through the flame (zero air) which will ionzie carbon. The ions are collected and measured as they create a current at the detectors electrode.

Question 33

what are the axis of a chromatgraph respresenting

Answer 33

X-axis: retention time of peak (tR), time the sample was injected into the column until it reaches the dector Y-axis: detector response: shows the measured response of the analyte peak within the detector

Question 34

what are the advantages and disadvanatages of gas chromotraghpy

Answer 34

1. high seperation effeciency 2. fast: ~ 20 minutes 3. small sample consumption and yet high detection sensitivity for 1 ul of sample 4. good selectivity disadvantages: it can only be used to analyze volatile substances. Would require substances to derived but this makes things messy and complex which can introduce errors

Question 35

describe HPLC

Answer 35

High-Performance Liquid Chromatography (HPLC) is used for separation, identification, and quantification of components in a mixture. It works by using high-pressure pumps to deliver a mixture of solvents (mobile phase) through a column packed with a solid material (stationary phase). The sample is injected and carried by the mobile phase through the column. As the components move through the column, they interact differently with the stationary phase—some bind more strongly and move slower, while others move faster. These differences in interaction result in different migration rates, allowing the components to be separated. The separated compounds are detected by instruments such as a UV detector.s

Question 36

what are the components of HPLC

Answer 36

1. pump 2. injector 3. column 4. dector

Question 37

what is a chromotraph

Answer 37

graphical represenation of signal strength as a function of time or volume, showing peaks that represent components of the sample