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Biochemistry And Endocrinology > Applications of Chromatography > Flashcards

Applications of Chromatography Flashcards

1
Q

what is an analyte?

A

Something we measure e.g drug, hormone, drug, electrolyte

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2
Q

Do immunoassays and other assays need to be sensitive?

A

yes

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3
Q

what are common assays?

A

Immunoassay, spectrophotometry, mass spectrometry

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4
Q

why would you use mass spectometry? - advantages

A

Used instead of immunoassay when selectivity of the antibody is not good.

It is selective, specific and sensitive

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5
Q

Disadvantages of mass spec?

A

slow
Expensive
Requires more training
Used in specilised and less accessible

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6
Q

Why would you use mass spec?

A

Identify a substance, modification, a metabolite
Quantify’s concentration and the rate of formation

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7
Q

What is precision?

A

How repeatable is this

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8
Q

What is accuracy?

A

How close to the correct answer is this

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9
Q

How would you compare assays?

A

Bland altman plot

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10
Q

Bland Altman Plot - how do you do this?

A

You plot the average of method 1 and 2 along the x-axis and the y-axis is the difference between the two assays - you want it to be 0 (you compare something you know gives you the right answer to a new thing)

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11
Q

in a bland altman plot what is bias and do you get this at low concentrations?

A

anything above 0 and yes

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12
Q

How is testosterone mesured?

A

Mass spec

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13
Q

Will you have to change the measurements of the assays dependant on gender, age and disease?

A

yes

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14
Q

what was the issues in a immunoassay of testosterone

A

other androgens where interfereing

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15
Q

what is the process of mass spec?

A

1) Separate out compound of interest from matrix
2) Vaporise the compound of interest to produce ions of interest
3) Separate ions based on their mass to charge ratio, m/z, where m = mass, z = charge

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16
Q

what is the purpose of seperation?

A

isolate it from other similar compounds

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17
Q

what are the main ways to seperate in mass spec?

A

1) Gas Chromatography-MS
2) Liquid Chromatography-MS

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18
Q

separation technique - chromatography

A

Chromatography is a physical method of separation in which the components to be separatedare distributed between two phases, one of which is stationary while the other moves in adefinite direction (mobile phase).

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19
Q

By what grounds are compounds seperated on chromatography?

A

molecular characteristics -size, volatility, ionisation, solubility

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20
Q

Gas chromatography Mass spec does splits things on what?

A

boiling point

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21
Q

How does gas chromatography mass spec work?

A

Inject substances into an injection port and as they boil they will flow through a column before going through a detector and a gas chromatogram - peaks show how much molecules are there

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22
Q

What does liquid chromatography mass spec separate on?

A

Achieved according to the partition coefficient of an analyte between solvents.

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23
Q

How do you calculate the partition coefficient?

A

Concentration of analyte in oily phase/concentration of analyte in water phase

24
Q

How do you do liquid chromatography mass spec?

A

Put substance on a column and pour liquid through, this will pull the substances through in a certain order and they are then put through a detector and this creates peaks.

25
Q

What is the backbone of the column?

A

Silica which can be modified to stick different compounds on top for more complicated mass spec.

26
Q

What are the types of ionisation?

A

1) volatile low molecular weight compounds which are thermally stable - electron impact or chemical ionisation
2) non-volatile, thermally label compounds - Electrospray (LC), atmospheric pressure chemical ionisation (LC)

27
Q

Electron impact - this is used in gas - how is this done?

A

You move ions from the filament to the anode and whilst they are travelling they get hit by electrons which knocks electrons and break bonds - this makes positive ions.

You can occasionally form negative ions.

28
Q

What is seen on the peaks after electron impact and why?

A

The ionised compounds must provide energy and thus the ions formed fragments to smaller ions - so lots of peaks

29
Q

What is the most abundant ion called and the molecular ion?

A

The most abundant ion is the base peak
Ion with mass of whole molecule = molecular ion

30
Q

Non-volatile thermally labilie compounds - electrospray - on what type of mass spec would you use this on?

A

Liquid

31
Q

How does an electrospray work?

A

Put the liquid into a charged capillary so the substance in the capillary is transferred charged. The liquid is evaporated and grows smaller which causes the molecules within the liquid to be unstable and explode - this then leads the molecule to have the positive or negative charge transferred onto it.

32
Q

Groups which charge more readily to form ions? Yes or no

A

Yes

33
Q

The electrospray peak?

A

These are simple, less fingerprints

34
Q

Does gas chromatography or liquid chromatography take longer

A

Yes

35
Q

What are the two main ways to detect the ions?

A

1) Beam - Magnetic sector, Time of flight, Quadropole
2) Traps - cyclotron

36
Q

How do ions get into the detector?

A

Charged plates - beams them into the analyser

37
Q

Quadrupole Mass analysers - how do these work?

A

The poles have radio frequency and charges meaning that when ions enter only one will have a stable trajectory - you can select for the drug or hormone you want.

38
Q

Pros of quadrupoles?

A

Low cost,
Bench top, multi user, robust
Ideal for quantition

39
Q

Cons of quadrupoles?

A

Limited mass range - can’t do proteins
Unit resolution
sensitivity drops and number of ions scanned increases

40
Q

Triple quadropole - the first one was previously described, what does the second one do?

A

Adds a collision gas

41
Q

Triple quadropole - what does the third one do?

A

Select for product

42
Q

Why would you use the triple quadropole? This can be called ms-ms

A

Helps with selectivity as some ions will give you the same number - you therefore have to put them through transitions.

43
Q

What are pros of ms-ms

A

Less back ground noise
Screens out interferences
Reduced sample prep times
Improved selectivity

44
Q

Cons of ms-ms?

A

Expensive
Staff training time

45
Q

What are reference methods?

A

Check amino assays or samples to see how much drugs there is

46
Q

What is an internal standard?

A

A molecule you put into every analysis to make sure you are doing it correctly

47
Q

How would you then quantify a drug with an internal standard?

A

Take the ratio

48
Q

What should your internal standard be?

A

A chemically similar to your sample as possible e.g. isotopomer, isomer, same chemical class

Ideally isotopomer

49
Q

What do you make to do you analyse - see if you are doing everything okay?

A

Make a standard curve by comparing internal standard and peak area and make it into a ratio (PLOT the RATIO).

50
Q

Case study vitamin D - why did we have issues with an immunoassay

A

There is lots of different types of vitamin Ds and its hard to tell them apart

51
Q

Quantitative analysis of concentration - what is therapeutic drug monitoring?

A

A way to measure drugs which have little therapeutic windows (they have to be monitored closely as they will go toxic with little prompt)

52
Q

Qualitative analysis is used to do what?

A

See what isn’t meant to be there

53
Q

Qualitative analysis - toxicology - how do they identify people taking drugs of abuse during sports?

A

Do a mass spec fingerprint (all the different peaks during gas chromatography mass spectrometry is this)

54
Q

How would you know if someone is abusing testosterone?

A

Measure the ratio of testosterone to epitestosterone if the ratio is higher than 6 = you’ve taken testosterone. But people would just take epitestosterone to keep this ratio correct.

People usually take testosterone from plants and you can compare plant and animal derived testosterone.

55
Q

What type of mass spectrometer do you use to compare plant and animal derived testosterone?

A

Isotope ratio mass spectrometer

This is boiled into carbon dioxide and you can measure the natural Animal testosterone and the plant testosterone carbon dioxide and compare it

56
Q

Qualitative analysis - inborn errors of metabolism - paediatric- what are the things which could be wrong and need qualitatively analysed?

A

steroid biosynthesis
Bile acid biosynthesis
Disorders of mitochondrial oxidation
Purines, pyrimidines, porphyria, catecholamine

57
Q

Why would you use mass spec on inborn errors of metabolism?

A

You need to know exactly what steroid they cannot metabolise in order to treat - you can look at peaks and see what’s being overproduced or underproduced.

Biochemistry And Endocrinology (16 decks)

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