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Chapter 25: Q17P (page 708)

(a) Nonpolar aromatic compounds were separated by HPLC on an octadecyl(C18)bonded phase. The eluent was 65 vol% methanol in water. How would the retention times be affected if 90% methanol were used instead?

(b) Octanoic acid and 1-aminooctane were passed through the same column described in (a), using an eluent of 20% methanol/80% buffer (pH 3.0). State which compound is expected to be eluted first and why.

role="math" localid="1656416023291" CH3CH2CH2CH2CH2CH2CH2CO2HOctanoicacidCH3CH2CH2CH2CH2CH2CH2CH2NH21鈥�Aminooctane

(c) Polar solutes were separated by hydrophilic interaction chromatography (HILIC) with a strongly polar bonded phase. How would retention times be affected if eluent were changed from 80 vol% to 90 vol% acetonitrile in water?

(d) Polar solutes were separated by normal-phase chromatographyon bare silica using methyl t-butyl ether and 2-propanol solvent. How would retention times be affected if eluent were changed from 40 vol% to 60 vol% 2-propanol? (Hint: See Table 25-4.)

Short Answer

Expert verified

(a) The non-polar compounds would be more soluble in the mobile phase.

(b) The 1-aminooctane would eluated first.

(c) The polarity of mobile phase decreases and the polar analytes are more strongly retained than non-polars.

(d) Less polar mobile phase.

Step by step solution

01

Step 1:The retention times be affected if 90% methanol

Part (a)

The retention time will be shorter if 90% methanol in water were used instead of 65%methanol in water because methanol is less polar than water.

So, the non-polar compounds would be more soluble in the mobile phase.

02

Step 2:Which compound is expected to be eluted first and why

Part (b)

At pH=3, the octanoic is in neutral form, while the 1-aminooctane is in the form of cation. Cation form is insoluble in the non-polar phase. So, if octanoic acid and 1- aminooctane passed through the same column using an eluent of 20% methanol/80% buffer (at pH 3.0), the 1-aminooctane would eluated first.

03

Step 3:The retention times be affected if eluent were changed from 80 vol% to 90 vol% acetonitrile in water

Part (c)

If polar solutes were separated by hydrophilic interaction chromatography (HILIC) with a strongly polar bonded phase, the retention time would be longer if eluent were changed from 80 % to 90 % acetonitrile in water because the polarity of mobile phase decreases and the polar analytes are more strongly retained than non-polars.

04

The retention times be affected if eluent were changed from 40 vol% to 60 vol% 2-propanol

Part (d)

If polar solutes were separated by normal-phase chromatography on bare silica (polar stationary phase) using methyl t-butyl ether and 2-propanol solvent (less polar mobile phase), the retention time would be shorter if eluent were changed from 40% to 60 % 2-propanol because 2-propanol has higher eluent strength than methyl t-butyl

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Most popular questions from this chapter

Question: Literature search problem: Human serum albumin (HSA) is an important protein ingredient in cryopreservation media used in procedures such as in vitro fertilization. Search the literature for a high-performance liquid chromatography method for the determination of human serum albumin and the stabilizer N-acetyl tryptophan in medical devices.

(a) Give the citation (authors, title, journal name, year, volume, pages) for the research paper that fits the criteria of this analysis.

(b) What alternative methods could be used for analysis of human serum albumin?

(c) What type of analytical column is used for the separation?

(d) How long was the gradient? How long were the additional wash and equilibration steps within the gradient method?

(e) What parameters were assessed in the method validation?

(f) Why were particles with 300 脜 pores used?

Question:The graph shows retention data from aC8silica column with an acetonitrile/water mobile phase.


(a) What mobile phase composition provides greatest retention(k)for the components? Least retention? Coelution (equalk)of two components?

(b) Predict the retention time of each peak at 40%and60%acetonitrite. Draw a chromatogram (a "stick diagram" representing each peak as a vertical line) of the separation at each mobile phase composition.

(c) Would60%acetonitrile yield adequate resolution?

(d) Assuming Gaussian peaks, does the separation at60%acetonitrile have the attributes of a good separation?

The rate at which heat is generated inside a chromatographycolumn from friction of flowing liquid is power (watts, W=J/s)= volume flow raterole="math" localid="1656474760665" (m3/s)脳pressure drop (pascals, role="math" localid="1656474828044" Pa=kg/[m?s2]).

(a) Explain the analogy between heat generated in a chromatography column and heat generated in an electric circuit (power = current脳voltage).

(b) At what rate (watts = J/s) is heat generated for a flow of 1 mL/minwith a pressure difference of3500 bar between the inlet and outlet?

You will need to convert mL/min tom3/s.Also 1 bar=105Pa.

(a) List ways in which the resolution between two closely spaced peaks might be changed.

(b) After optimization of an isocratic elution with several solvents, the resolution of two peaks is 1.2How might you improve the resolution without changing solvents or the kind of stationary phase?

(a) You wish to use a wide gradient from 5 vol % to 95 vol %B for the first separation of a mixture of small molecules to decide whether to use gradient or isocratic elution. What should be the gradient time, tG,for a 15脳0.46-cmcolumn containing 3-渭尘particles with a flow of 1.0mL/min

You optimized the gradient separation going from 20vol%to 34 vol% Bin 11.5min at 1.0mLFindk*for this optimized

separation. To scale up to a 15脳1.0cmcolumn, what should be the gradient time and the volume flow rate? If the sample load on

the small column was 1mgwhat sample load can be applied to the large column? Verify that k*is unchanged.
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