Chapter 2 - Basic terms and concepts - part 2

Question 1

Ideally, solvents used as HPLC mobile phase should have what characteristics?

Answer 1

• Good solubility of sample components
• Noncorrosive to HPLC
• High purity, low cost, UV transparent
• Low viscosity, toxicity, and nonflammability

Question 2

Solvent strength refers to

Answer 2

the ability of a solvent to elute solutes from a column.

Question 3

Solvent strength is related to its

Answer 3

polarity.

Question 4

ACN is the preferred RPC solvent because of its

Answer 4

• low viscosity
• higher column efficiency
• good UV transparency
• strong elution strength

Question 5

The modern trend is to use buffer strengths between

Answer 5

5-20 mM.

Question 6

In RPC, a pH of ______ is used for many applications.

Answer 6

2-4

Question 7

Most silica-based bonded phases are not stable below pH

Answer 7

2

Question 8

Ion-pairing reagents are

Answer 8

detergent-like molecules added to the mobile phase to provide retention of acidic or basic analytes.

Question 9

Ion-pairing reagents use

Answer 9

long-chain alkyl sulfonates to combine with basic solutes under acidic pH to form neutral “ion pairs” which are retained in RPC.

Question 10

Typical flow rates for analytical columns are

Answer 10

0.5-2 ml/min.

Question 11

Operating flow rates are proportional to

Answer 11

the square of the column inner diameter.

Question 12

The degree of separation or resolution (R_s) between two solutes is dependent on both

Answer 12

thermodynamic factors (k and alpha) and kinetic factors (w_b and N)

Question 13

Under isocratic conditions, resolution is controlled by three factors

Answer 13

1. retention
2. selectivity
3. efficiency

Question 14

Doubling N by doubling the column length increases analysis time by a factor of _____ but increases resolution by a factor of

Answer 14

2; √2

Question 15

Sequential isocratic steps

Answer 15

A type of method development approach that works by lowering the solvent strength (e.g. 60% ACN to 40% ACN).

Question 16

Elution strength of ACN, MeOH, and THF if RPC.

Answer 16

MeOH < ACN < THF

Question 17

Typical Van Deemter curve labels

Answer 17

HETP vs. V

Question 18

The dip, or minimum point on the Van Deemter curve, marks

Answer 18

the minimum plate height (H_min) found at the optimum flow velocity (V_opt)

Question 19

Van Deemter equation

Answer 19

HETP = A + B/V + CV

Question 20

The term A in the Van Deemter equation is approximately equal to ______ in well packed columns.

Answer 20

2d_p

Question 21

The B term in the Van Deemter equation is proportional to

Answer 21

D_m of the solute.

Question 22

The contribution from the B term in the Van Deemter equation is only important at

Answer 22

very low flow rates.

Question 23

The C term in the Van Deemter equation is proportional to

Answer 23

d_p²/D_m

Question 24

The C term in the Van Deemter equation becomes significant at

Answer 24

high flow rates for columns packed with larger particles.

Question 25

Small d_p yields _____ H_min.

Answer 25

small

Question 26

The H_min for a 3 μm particle column is approximately

Answer 26

6 μm.

Question 27

Small-particle columns suffer _______ efficiency loss at high flow rates.

Answer 27

less

Question 28

UHPLC equipment must be used for columns with particles under how many μm?

Answer 28

2 μm.

Question 29

Advantages of gradient analysis (5)

Answer 29

• 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.

Question 30

Disadvantages of gradient analysis. (4)

Answer 30

• 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.

Question 31

Isocratic peaks will _______ with elution time.

Answer 31

broaden

Question 32

Peak capacity is a useful concept for

Answer 32

comparing column performance under different gradient conditions.

Question 33

Peak capacity is the

Answer 33

maximum number of peaks that can fit in a chromatogram with a resolution value of one.

Question 34

Peak capacity equation

Answer 34

n = 1 + t_g/w_b

Question 35

The “0.25 Δt_G Rule” is used to

Answer 35

determine whether a sample run under gradient conditions can be more effectively handled by isocratic analysis.

Question 36

Orthogonal separation method

Answer 36

Using an alternative method to ensure no impurities are coeluting with analytes.

Question 37

For orthogonal separation, a method that produces similar or different results should be used?

Answer 37

Different

Question 38

Sample capacity is

Answer 38

the maximum amount of solute in milligrams per gram of packing material that can be injected without a significant reduction in column efficiency.