HPLC Flashcards
HPLC Diagram
mp
Pump
Injector
Column
Detector
Purpose of mobile phase
Move sample through system and pass the stationary phase
MP in HPLC
Always a liquid
eg methanol and water 70:30
Has ideal characteristics
MP in HPLC ideal characteristics
Shouldn’t interact with sample
High purity
Readily available
Compatible with detector
Purpose of the pump in HPLC
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
Note on the injector in HPLC
Is a syringe and needle
Fully automated, based on a router that switches between load and inject positions
GC : one ul
HPLC : 10 ul
Column used in HPLC
Two types the guard and analytical column
Analytical column in HPLC
Contains SP eg C18
15cm long and made of steel
Where does separation occur in HPLC
In the analytical column
Why would you use small SP particles in HPLC and give a disadvantage
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
What u is s a guard column?
Short column that’s attached to analytical column, only 1-2cm long
Is changed regularly
Why is a guard column used
Used to protect the analytical column as it absorbs any impurities that could damage the analytical column
Detector used in HPLC
UV-Vis detector
Purpose of UV-Vis detector
It can measure absorbance. Abs is the area under the curve and is proportional to concentration
What happens in HPLC
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.
Normal phase
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
Reverse phase
Used in HPLC
SP non polar eg C18
MP polar eg water and methanol 70:30
Analytes: caffeine
Generally separating polar components
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
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
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)
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.
If you make the mp more non polar in HPLC with polar analytes
This will increase retention times
How to decrease retention times using the mp
Make mp more like sample
Effects of making mp more like the sample
Better separation of sample
Shorter run times
Able to deal with complex mixtures
The types of solvent systems in HPLC
Isocratic elution and gradient elution
An isocratic system
Holds the composition of mp constant eg always running at 70:30
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
Benefits of using a gradient elution system
Same benefits as using temperature programming in GC
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.
Types of UV vis detectors
Fixed wavelength and photo diode array
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
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
Characteristics of a good detector in HPLC
Same as the characteristics in GC
Name the device that is used to collect separate portions that elute
Fraction detector can collect at a set time or a set volume
MP and SP in HPLC
mp liquid pumped through column polar
sp liquid coated on the column eg c18 non polar
What does two peaks on a chromatogram indicate
Two compounds in the sample
What does the sharpness of the peak represent
The efficiency
What does resolution mean
How well separated the plates are
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
Adequately resolved peaks
Has a resolution greater than one
Conditions that affect the quality of separation
PARAMETRES
Flow rates
Type of column
Type of mobile phase
Most popular, powerful and versatile form of chromatography
HPLC
HPLC mode
adsorption, partition, ion exchange or size exclusion
HPLC mobile phase and stationary phase
Mobile Phase = liquid (pumped by high pressure)
Stationary Phase = liquid chemically bonded to the column packing (eg, C18)
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
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
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
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
Which column achieves the separation in HPLC
The analytical column
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
Detectors used for HPLC
UV-Visible Absorbance Detector
(This is a miniature spectrophotometer)
Either a fixed wavelength detector or photo diode array
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
Photo diode array uv vis detector advantages
Advantages:
better identification
choose best wavelength for each analyte much faster
Mass spec detector
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
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
Slide
mode summary
packed vs capillary columns
Fid
Rey time
Advantages of Capillary Columns
Extremely good resolution
Separate complex mixtures
Separate similar compounds, eg isomers Very narrow peaks
Low detection limits
Problems with GLC
Analyte sometimes not volatile – convert to volatile derivative, chemical reaction
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
Qualitative uses of chromatography
Spiking
Mass Spectrometer as detector
Compare Retention Time to known standards in the same conditions
External Standard Problem
Injection size inconsistent
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)
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
Resolution
Twice the difference in retention times / the sum of base width
Efficiency formula
N = 5.5 ( retention time / peak width at half height ) squared
N = 16 ( retention time / base width) squared
measured in plates
Number of plates per metre
Number of plates per m = N / divided by length in metres
