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Analytical > HPLC > Flashcards

HPLC Flashcards

1
Q

HPLC Diagram

A

mp
Pump
Injector
Column
Detector

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2
Q

Purpose of mobile phase

A

Move sample through system and pass the stationary phase

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3
Q

MP in HPLC

A

Always a liquid
eg methanol and water 70:30
Has ideal characteristics

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4
Q

MP in HPLC ideal characteristics

A

Shouldn’t interact with sample
High purity
Readily available
Compatible with detector

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5
Q

Purpose of the pump in HPLC

A

Pushes everything through the system by operating at a high pressure (1000 psi)
High pressure required to overcome back pressure from column
Flow rate 1-10ml/minute

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6
Q

Note on the injector in HPLC

A

Is a syringe and needle
Fully automated, based on a router that switches between load and inject positions

GC : one ul
HPLC : 10 ul

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7
Q

Column used in HPLC

A

Two types the guard and analytical column

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8
Q

Analytical column in HPLC

A

Contains SP eg C18
15cm long and made of steel

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9
Q

Where does separation occur in HPLC

A

In the analytical column

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10
Q

Why would you use small SP particles in HPLC and give a disadvantage

A

Increase the surface area of SP which means more packing of particles inside the column.
Disadvantage: a higher pressure is needed to push everything through the smaller particles

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11
Q

What u is s a guard column?

A

Short column that’s attached to analytical column, only 1-2cm long
Is changed regularly

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12
Q

Why is a guard column used

A

Used to protect the analytical column as it absorbs any impurities that could damage the analytical column

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13
Q

Detector used in HPLC

A

UV-Vis detector

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14
Q

Purpose of UV-Vis detector

A

It can measure absorbance. Abs is the area under the curve and is proportional to concentration

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15
Q

What happens in HPLC

A

Sample gets injected at the injector is pushed through by mp which is pumped by the pump, separation happens with the sp in the analytical column, peaks are detected on uv vis detector.

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16
Q

Normal phase

A

Used in TLC and GC
SP polar eg silica
MP non polar eg petroleum ether and acetone 70:30 ratio
Analytes: spinach pigments
Generally separating non polar components

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17
Q

Reverse phase

A

Used in HPLC

SP non polar eg C18
MP polar eg water and methanol 70:30
Analytes: caffeine
Generally separating polar components

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18
Q

2 compounds x and y elute from a normal phase column in that order. Explain why they’re likely to elute in the Oder y and x in a reverse column

A

Normal phase
X first and Y second
Polar SP:
Y is more polar and will have a longer retention time as it has a higher affinity for the stationary phase

Reverse phase
Y first and X second
Non polar SP
X is more non polar and will have a longer retention time as it has a higher affinity for the stationary phase

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19
Q

Describe how elution times of analytes are changed by changing the polarity of the mobile phase in reverse phase.
(In reverse phase how can you change the retention times)

A

If you make the mp more like the compounds they will elute quicker and reduce the retention times of A and B to two and five minutes.

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20
Q

If you make the mp more non polar in HPLC with polar analytes

A

This will increase retention times

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21
Q

How to decrease retention times using the mp

A

Make mp more like sample

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22
Q

Effects of making mp more like the sample

A

Better separation of sample
Shorter run times
Able to deal with complex mixtures

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23
Q

The types of solvent systems in HPLC

A

Isocratic elution and gradient elution

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24
Q

An isocratic system

A

Holds the composition of mp constant eg always running at 70:30

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25
Q

Gradient elution system

A

Changing the composition of the mp as the experiment continues
eg begin with water at 40% then gradually ramp up to 70% water

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26
Q

Benefits of using a gradient elution system

A

Same benefits as using temperature programming in GC

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27
Q

The importance of the elutropic series

A

The index ranks solvents in terms of polarity and allows you to make decisions around changing the mobile phase.
As you go down the table polarity increases.

28
Q

Types of UV vis detectors

A

Fixed wavelength and photo diode array

29
Q

Fixed wavelength UV vis detector

A

Measures absorbance at only one wavelength
Cheap and easy to use
However can’t see two compounds abs at once

30
Q

Photo diode array detector

A

Measures more than one wavelength at the same time

Creates a 3D graph of time v wavelength v absorbance
More expensive
More complex engineering

eg caffeine adsorbs at 273nm

Measures abs at lots of times and lots of wavelengths

31
Q

Characteristics of a good detector in HPLC

A

Same as the characteristics in GC

32
Q

Name the device that is used to collect separate portions that elute

A

Fraction detector can collect at a set time or a set volume

33
Q

MP and SP in HPLC

A

mp liquid pumped through column polar
sp liquid coated on the column eg c18 non polar

34
Q

What does two peaks on a chromatogram indicate

A

Two compounds in the sample

35
Q

What does the sharpness of the peak represent

A

The efficiency

36
Q

What does resolution mean

A

How well separated the plates are

37
Q

Efficiency

A

Column plates stacked on top of one another
n is the number of theoretical plates
Sample partitions between plates and mp
The more plates the better the efficiency

38
Q

Adequately resolved peaks

A

Has a resolution greater than one

39
Q

Conditions that affect the quality of separation

A

PARAMETRES
Flow rates
Type of column
Type of mobile phase

40
Q

Most popular, powerful and versatile form of chromatography

A

HPLC

41
Q

HPLC mode

A

adsorption, partition, ion exchange or size exclusion

42
Q

HPLC mobile phase and stationary phase

A

Mobile Phase = liquid (pumped by high pressure)
Stationary Phase = liquid chemically bonded to the column packing (eg, C18)

43
Q

Properties of a good mobile phase in HPLC

A

High purity

Readily available

A boiling point 20 – 50oC above the

column temp Low viscosity

Low reactivity
Compatibility with the detector

44
Q

Types of analysis HPLC

A

Isocratic analysis

The same mobile phase is used throughout the separation

Gradient Elution analysis

Two (or more) different solvents combined, the relative amounts of each altered throughout the separation. The gradient can be
Continuous
Stepped
The gradient elution is controlled by a computer

45
Q

How to adjust the polarity of the mobile phase in HPLC

A

Use the eluotropic series which list possible solvents in terms of their polarity

Eg cyclohexane is at the top of the series and is non polar and water is at the bottom because it is the most polar

46
Q

Pump in HPLC

A

delivers mobile phase at between 0.1 and 10 ml/min Pressure must overcome the backpressure of the column
1000 - 5000 psi

47
Q

Which column achieves the separation in HPLC

A

The analytical column

48
Q

Ideal detector in HPLC

SWLLGDF

A

An ideal detector for HPLC (like GLC) would have:
Similar response to all kinds of compounds
Wide concentration range
Low detection limit
Linear response
Good resolution
Fast response to change Stability
Reliability
Ease of use
And be non-des

49
Q

Detectors used for HPLC

A

UV-Visible Absorbance Detector

(This is a miniature spectrophotometer)

Either a fixed wavelength detector or photo diode array

50
Q

Fixed wavelength uv vis v photo diode array uv vis detector

A

Fixed wavelength (Problem - two components of a mixture may not absorb at the same wavelength

Photo-diode Array
Plots a complete absorbance spectrum

51
Q

Photo diode array uv vis detector advantages

A

Advantages:
better identification
choose best wavelength for each analyte much faster

52
Q

Mass spec detector

A
53
Q

Ion exchange chromatography

sp
mp
interactions
used in
Uses

A

SP = Polymer + anions (SO3 -) or cations (N(CH3)3+)
MP = ionic solution
Electrostatic attraction Column, HPLC
Drug analysis and protein separation

54
Q

Size Exclusion Chromatography
sp
mp
separation depends on:
uses

A

SP = porous gel
MP = liquid
Size :

Large molecules – elute first will be the first peak on a chromaogram
Very small molecules – elute last will be the laT peak on a chromatogram

Column, HPLC

Uses:
Size Exclusion Uses
Gel filtration chromatography – Used for separation of proteins and other water-soluble large molecules such as polysaccharides and nucleic acids

55
Q

Slide

mode summary
packed vs capillary columns
Fid
Rey time

A
56
Q

Advantages of Capillary Columns

A

Extremely good resolution
Separate complex mixtures
Separate similar compounds, eg isomers Very narrow peaks
Low detection limits

57
Q

Problems with GLC

A

Analyte sometimes not volatile – convert to volatile derivative, chemical reaction

58
Q

Applications of HPLC

A

Quantification of pharmaceutical products
Determination of food additives, eg growth promoters and vitamins
Determination of herbicide and pesticide residues Analysis of clinical samples

59
Q

Qualitative uses of chromatography

A

Spiking
Mass Spectrometer as detector
Compare Retention Time to known standards in the same conditions

60
Q

External Standard Problem

A

Injection size inconsistent

61
Q

Internal Standard
M P N S

A

Must be miscible with the sample solution
Must be pure
Must not be in the sample normally
Must be stable and non-reactive
Must be structurally similar to the analytes
Must be chromatographically separable from all other components
Must elute from the chromatographic column close to the analyte(s)

62
Q

Retention factor k

A

RT divided by RT of solvent

High retention factor –
high retention time –
good resolution But not too high – best between 1 and 5

63
Q

Resolution

A

Twice the difference in retention times / the sum of base width

64
Q

Efficiency formula

A

N = 5.5 ( retention time / peak width at half height ) squared

N = 16 ( retention time / base width) squared

measured in plates

65
Q

Number of plates per metre

A

Number of plates per m = N / divided by length in metres

Analytical (9 decks)

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