Chapter 2 - Basic terms and concepts - part 2 Flashcards
Ideally, solvents used as HPLC mobile phase should have what characteristics?
- Good solubility of sample components
- Noncorrosive to HPLC
- High purity, low cost, UV transparent
- Low viscosity, toxicity, and nonflammability
Solvent strength refers to
the ability of a solvent to elute solutes from a column.
Solvent strength is related to its
polarity.
ACN is the preferred RPC solvent because of its
- low viscosity
- higher column efficiency
- good UV transparency
- strong elution strength
The modern trend is to use buffer strengths between
5-20 mM.
In RPC, a pH of ______ is used for many applications.
2-4
Most silica-based bonded phases are not stable below pH
2
Ion-pairing reagents are
detergent-like molecules added to the mobile phase to provide retention of acidic or basic analytes.
Ion-pairing reagents use
long-chain alkyl sulfonates to combine with basic solutes under acidic pH to form neutral “ion pairs” which are retained in RPC.
Typical flow rates for analytical columns are
0.5-2 ml/min.
Operating flow rates are proportional to
the square of the column inner diameter.
The degree of separation or resolution (Rs) between two solutes is dependent on both
thermodynamic factors (k and alpha) and kinetic factors (wb and N)
Under isocratic conditions, resolution is controlled by three factors
- retention
- selectivity
- efficiency
Doubling N by doubling the column length increases analysis time by a factor of _____ but increases resolution by a factor of
2; √2
Sequential isocratic steps
A type of method development approach that works by lowering the solvent strength (e.g. 60% ACN to 40% ACN).
Elution strength of ACN, MeOH, and THF if RPC.
MeOH < ACN < THF
Typical Van Deemter curve labels
HETP vs. V
The dip, or minimum point on the Van Deemter curve, marks
the minimum plate height (Hmin) found at the optimum flow velocity (Vopt)
Van Deemter equation
HETP = A + B/V + CV
The term A in the Van Deemter equation is approximately equal to ______ in well packed columns.
2dp
The B term in the Van Deemter equation is proportional to
Dm of the solute.
The contribution from the B term in the Van Deemter equation is only important at
very low flow rates.
The C term in the Van Deemter equation is proportional to
dp2/Dm
The C term in the Van Deemter equation becomes significant at
high flow rates for columns packed with larger particles.
Small dp yields _____ Hmin.
small
The Hmin for a 3 μm particle column is approximately
6 μm.
Small-particle columns suffer _______ efficiency loss at high flow rates.
less
UHPLC equipment must be used for columns with particles under how many μm?
2 μm.
Advantages of gradient analysis (5)
- Better suited for complex samples that require quantitation of all peaks.
- Better resolution of early and late eluting peaks.
- Peaks have similar widths throughout the entire run.
- Better sensitivity for late eluting peaks.
- Higher peak capacity.
Disadvantages of gradient analysis. (4)
- More complex HPLC instrument is required.
- Gradient methods are more difficult and time-consuming to develop.
- Transfer of gradient methods from one lab to another is more difficult.
- Longer run times due to column equilibration.
Isocratic peaks will _______ with elution time.
broaden
Peak capacity is a useful concept for
comparing column performance under different gradient conditions.
Peak capacity is the
maximum number of peaks that can fit in a chromatogram with a resolution value of one.
Peak capacity equation
n = 1 + tg/wb
The “0.25 ΔtG Rule” is used to
determine whether a sample run under gradient conditions can be more effectively handled by isocratic analysis.
Orthogonal separation method
Using an alternative method to ensure no impurities are coeluting with analytes.
For orthogonal separation, a method that produces similar or different results should be used?
Different
Sample capacity is
the maximum amount of solute in milligrams per gram of packing material that can be injected without a significant reduction in column efficiency.
