360 - Chromatography Flashcards
What is the mobile phase
a liquid or gas that carriers the sample through the stationary phase
What is the stationary phase
a solid or liquid which interacts with the sample and the mobile phase
Chromatography can be separated into 2 groups -
planar and columnar based on the geometric shape of the support material.
Planar chromatography
the stationary phase is supported by a flat surface such as a glass plate
Many POCT dry slide tests use the principle of planar chromatography
Thin layer chromatography is used in the biochemical genetics labs to detect inherited disorders of amino acid metabolism
Column chromatography
the stationary phase is supported by a column
can be made of materials such as glass and stainless steel
two groups - based on the type of mobile phase used
- liquid chromatography (LC) the mobile phase is a liquid while the stationary phase may be a solid or another liquid
- gas chromatography, the mobile phase is a gas while the stationary phase is usually a liquid
Chromatogram
a plot of the detector response as a function of the time or volume of the mobile phase that is needed to elute a compound from a column
Retention time (tR)
the interval between specimen injection and the solute reaching the detector; retention time helps identify an analyte
Retention volume (VR)
the volume of eluent carrier gas admitted to the column between the injection of the sample and the emergence of the peak maximum of the specified component
How can analyzes be identified using a chromatogram?
Analytes can be identified by comparing their retention time or retention volume to a known analyte separated under thee same conditions
NOTE: In order to identify an analyte the peaks of the analyte peak must be separated or solved from adjacent peaks. The measurement of this separation is the resolution.
Resolution (Rs)
a measurement of the separation of two neighbouring peaks; it is calculated by subtracting the earlier peak retention time or volume from the later peak and dividing the by the average baseline peak widths (Wb) of the two peaks
T or F. The larger the resolution the greater the separation between the two peaks
T! Ideally, a Rsvalue of 1.5 or greater indicates complete separation of the two peaks
T or F. The goal is to have the detector signal return to baseline in between peaks
T, however as the resolution increases the amount of time or volume of mobile phase used to separate the two analytes increases, this will affect turnaround time
Peak resolution is influenced by the …
efficiency of the column, an analyte’s retention factor (k), and the separation factor (α) for two analytes
Efficiency
a measure of chromatographic performance that usually is related to the sharpness of peaks of a chromatogram
Theoretical plate #
- efficiency of a column is defined by this (N)
- the number of times an analyte transitions from the mobile phase to the stationary phase and back to the mobile phase
- the greater the number of these steps the easier it is to separate two analytes with similar retention times or volumes
T or F. Decreasing the length of a column will increase efficiency
F! Increasing the length
Efficiency can be manipulated by …
Increasing length of column
altering the flow rate of the mobile phase
using a smaller diameter support material or smaller diameter column e.g. capillary column
Why do we NOT want analytes to spend a long time in a column
The longer it spends there, the more it will diffuse = wider bands
Retention factor (k)
a measure of retention in the chromatograph; it is the ratio of the volumes of the mobile and stationary phase in the column
Small k vs large k value
a small k value indicates the analyte is poorly retained by the column
a large k value indicates the analyte is retained by the column and longer analysis time is required
How is the retention factor calculated?
retention factor is calculated by subtracting the void time or volume from the analyte’s retention time or volume divided by the void time or volume
a measure of the degree of separation
Separation factor (alpha)
equal to the ratio of the retention factors for two adjacent peaks, where the larger retention factor (k) is the numerator
Separation factor relationship with retention factor
If two analytes have the same (equal) retention factor, the separation factor is one. As the value of the separation factor increases (>1) the degree of separation between the two analytes increases
T or F. peak area calculations are more accurate than peak heights
T!
External calibration/ external std technique
a set of standards of known concentration are processed and run with the sample
standard curve (calibration curve) is generated and the sample peak is compared to the curve to calculate the concentration
Internal calibration/ internal std technique
a compound, the internal standard, is added in a known concentration to both the external standards and sample
standards and sample are processed and analyzed in the same run
calibration curve is generated by plotting the ratio of external standard to internal standard [this curve is used to determine the concentration of the analyte]
This technique helps normalizes any variation caused by pre-treatment steps
internal standards must be pure and of a known concentrations; must not normally be found in the sample and should elute close to the analyte
T or F. The SA of the particle in packed columns is inversely proportional to pore diameter
T!
types of SEC
classified by mobile hase
- aqueous = gel filtration chromatography
- organic =gel permeation chromatography
most common type of high pressure pump
dual piston reciprocating pump
- product pulsation in moile phase
problem caused by pulsation
can increase baseline signal
- solve by pulse dampers
guard column
between injector and analytical column; protects it
trap particulate matter and strongly retained sample components
bind components that would irreversibly bind to analytical column
column
before analytical column
- saturates mobile phase w silicates
= conditions mobile phase before main HPLC column
different types of HPLC detectors
- absorbance = compound w chromophores absorb UV or visible light
- fluorescence = compounds w fluorophores
- electrochemical = redox rxns
- conductivity = ionic solutes
- refractive index = universal detector
- mass spec = universal in full scan mode or selective ion monitoring mode
these detectors are commonly used in HPLC
absorbance dectectors
advantages of gradient elution method
retained sample components eluted quicker
reduced separation time
decreases band broadening
inlet filters
on solvent reservoir lines
can filter out particulates
As the size of particle in liquid chromatogaphy decreases, the resolution _______ but the pressure _____________.
resolution increases
pressure also increases
pre-column
placed before columns w silica support particles to prevent silica particle from dissolving in the mobile phase
packed w silica-based particles; saturates mobile phase w silicates
