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HPLC Flashcards

(65 cards)

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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
Gradient elution system
Changing the composition of the mp as the experiment continues eg begin with water at 40% then gradually ramp up to 70% water
26
Benefits of using a gradient elution system
Same benefits as using temperature programming in GC
27
The importance of the elutropic series
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
Types of UV vis detectors
Fixed wavelength and photo diode array
29
Fixed wavelength UV vis detector
Measures absorbance at only one wavelength Cheap and easy to use However can’t see two compounds abs at once
30
Photo diode array detector
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
Characteristics of a good detector in HPLC
Same as the characteristics in GC
32
Name the device that is used to collect separate portions that elute
Fraction detector can collect at a set time or a set volume
33
MP and SP in HPLC
mp liquid pumped through column polar sp liquid coated on the column eg c18 non polar
34
What does two peaks on a chromatogram indicate
Two compounds in the sample
35
What does the sharpness of the peak represent
The efficiency
36
What does resolution mean
How well separated the plates are
37
Efficiency
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
Adequately resolved peaks
Has a resolution greater than one
39
Conditions that affect the quality of separation
PARAMETRES Flow rates Type of column Type of mobile phase
40
Most popular, powerful and versatile form of chromatography
HPLC
41
HPLC mode
adsorption, partition, ion exchange or size exclusion
42
HPLC mobile phase and stationary phase
Mobile Phase = liquid (pumped by high pressure) Stationary Phase = liquid chemically bonded to the column packing (eg, C18)
43
Properties of a good mobile phase in HPLC
High purity Readily available A boiling point 20 – 50oC above the column temp Low viscosity Low reactivity Compatibility with the detector
44
Types of analysis HPLC
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
How to adjust the polarity of the mobile phase in HPLC
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
Pump in HPLC
delivers mobile phase at between 0.1 and 10 ml/min Pressure must overcome the backpressure of the column 1000 - 5000 psi
47
Which column achieves the separation in HPLC
The analytical column
48
Ideal detector in HPLC SWLLGDF
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
Detectors used for HPLC
UV-Visible Absorbance Detector (This is a miniature spectrophotometer) Either a fixed wavelength detector or photo diode array
50
Fixed wavelength uv vis v photo diode array uv vis detector
Fixed wavelength (Problem - two components of a mixture may not absorb at the same wavelength Photo-diode Array Plots a complete absorbance spectrum
51
Photo diode array uv vis detector advantages
Advantages: better identification choose best wavelength for each analyte much faster
52
Mass spec detector
53
Ion exchange chromatography sp mp interactions used in Uses
SP = Polymer + anions (SO3 -) or cations (N(CH3)3+) MP = ionic solution Electrostatic attraction Column, HPLC Drug analysis and protein separation
54
Size Exclusion Chromatography sp mp separation depends on: uses
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
Slide mode summary packed vs capillary columns Fid Rey time
56
Advantages of Capillary Columns
Extremely good resolution Separate complex mixtures Separate similar compounds, eg isomers Very narrow peaks Low detection limits
57
Problems with GLC
Analyte sometimes not volatile – convert to volatile derivative, chemical reaction
58
Applications of HPLC
Quantification of pharmaceutical products Determination of food additives, eg growth promoters and vitamins Determination of herbicide and pesticide residues Analysis of clinical samples
59
Qualitative uses of chromatography
Spiking Mass Spectrometer as detector Compare Retention Time to known standards in the same conditions
60
External Standard Problem
Injection size inconsistent
61
Internal Standard M P N S
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
Retention factor k
RT divided by RT of solvent High retention factor – high retention time – good resolution But not too high – best between 1 and 5
63
Resolution
Twice the difference in retention times / the sum of base width
64
Efficiency formula
N = 5.5 ( retention time / peak width at half height ) squared N = 16 ( retention time / base width) squared measured in plates
65
Number of plates per metre
Number of plates per m = N / divided by length in metres

Analytical (9 decks)

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