Lecture 4: Chromatography Terminology And Gas Chromatograhy Flashcards
Chromatogram ( the graph with detector response against time )
Qualitative
- retention time (Rt) same under identical chromatographic conditions (however should use mass spec to back claim)
Quantitative
- peak area is proportional to sample concentration. Need a standard curve ie the higher the concentration the broader the peak
Semi-quantative
This peak is bigger than that peak therefore higher conc
Basic terminology: retention time (tR), t₀ and Wb
tR - time from injection to appearance of peak maximum
t0 - retention time of an unretained solute (when material does not interact with the stationary phase and is stuck in the mobile phase so goes straight through)
Wb - base width of peak
How the peak should look
Want symmetrical (Gaussian) and sharp peak
Wb becomes larger with longer tR
Temp affects retention time
Basic terminology: retention or capacity factor (k’)0
It is a measure of peak retention (ie the peak of the peak and so tells you how much time analyt spends on column to compared to t0)
How many times the peak is retained vs unretained peak (kind of like Rf value in TLC)
k’ = 0 at t₀, k’ = 1 at 2t0, k’ = 2 at 3t₀ etc
k’ = ( tR - t₀ ) / t₀
Seperation or selectivity factor (alpha)
Measure of the separation of peaks
Ratio of differential retention of two analyses by the column
a must be > 1.0 for peak separation (ideally larger)
a = k’₂ / k’₁
Column efficiency N
This is the number of theoretical plates
Ie calculating how tall and narrow the peaks are
Ie for a resolution of 1.5 the number of theoretical plates is super high. This shows the peak is sharp with good seperation between peaks (high efficiency)
For a resolution of 0.8 there are a lot less plates. It is low efficiency (broader peak, poor seperation)
Separation equilibrations of the sample between the stationary and mobile phase occurs in these “plates”. The anaylate moves down the column by transfer of wquilibrated mobile phase from one plate to the next
Column plate number (N)
It is a measure of column efficiency
Height equivalent of theoretical plate (HETP) or plate height (H) measures of column efficiency per unit length (L) of column
N = L / H or N = L / HETP
Column is efficient when HETP is small and N is large
N = 16 ( tR / Wb )^2
Resolution (Rₛ)
Rs is a measure of the degree of seperation of two peaks
If R/s = 0 no separation, Rs = 1 partial separation, Rs = 1.5 baseline separation (ie Rs should be >2)
Rs = 2( tR₂-tR₁ ) / ( Wb₁ + Wb₂ )
Resolution represents the separation power of the whole system
Resolution equation - good graph on slide 10
Rs = 1/4 √(N) × (a - 1) / (a) × (k’) / (1 + k’)
Efficiency selectivity Retention (between 2-10)
What to look out for in chromatogram
Baseline seperation / no co-elution
Peak tailing / symmetry
Eluting not too soon or too late
Response ok?
Got the amount of peaks expected ie compounds expected
Gas chromatography (GC)
Liquid sample goes into sample injection unit
Each component is heated and vaporised
In column:
Seperation of components
Detector:
Convert the amount of each component into an electrical signal
Gas Chromatography is an analytical separation technique in which a mixture of volatile chemical constituents are vaporised and then resolved by the partitioning of the analytes carried by an inert gas over a stationary phase and detected either for qualitative or qualitative purpose
when can GC be used?
Used in analysis of compounds which are volatile below 300°C and are thermally stable → x want decomposition
Usually molecules MW<500 Da
NOT high MW materials - polymers, peptides etc. (small molecular masses)
Materials can be derivatised to aid volatility
Very small quantities required (roughly 1 mm of sample)
GC advantages
• Fast
• Efficient - high resolution
• Sensitive - ppm
• Non-destructive - hyphenated technique
• Quantitative - peat area is prop to conc
• Small sample
• Reliable and simple
• Cheap
GC disadvantages
• Limited to volatile samples
• Unsuitable for thermally labile samples
• Requires spectroscopy (usually MS) for identification of peak identity
• Not suited for preparative chromatography
• Only ~20% of known organic compounds can be analysed by GC
GC apparatus - slide 19
Carrier gas ( H2, He, N2) - mobile phase pumped through flow controller.
This enters a place where the sample is injected into and then both travel through a coil (v long and thin) called the column until it reaches the detector at the end. The place, column and detector are all in an oven.
Detector attaches to an amplifier which helps produce the Chromatogram of U (detection response - y axis) and Rt (retention time on x axis)
Carrier gas
This is the mobile phase
Must be inert - can’t react with sample, high purity and dry
Must be specific for detector type
Carries sample into and through the column
Provides suitable matrix for the detector to measure sample components
He - compatible w mass spec BUT is finite and expensive
N2 - cheap and safe BUT viscous so have to push it through slowly
H2 - reactive
What does the Van Demeter Plot show you
Column efficiency HETP (mm) on y axis against flow rate (carrier gas velocity cm/s)
The lower the HETP the better - Dip shows the optimum linear velocity
H2 gives the best range
Sample introduction
• Introduce sample as sharp symmetrical bands
• Representative sample
• No chemical change
• Repeatable and reproducible
• Injection techniques
- Splitless (SL)
- Split (S)
- Large volume injection (LVI)
- On-column
Typical injector - slide 28
Look at diagram
