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Chapter 25: Q37P (page 711)

A reversed-phase separation of a reaction mixture calls for isocratic elution with 48%methanol 52%water. If you want to change the procedure to use acetonitrile/water, what is a good starting percentage of acetonitrile to try?

Short Answer

Expert verified

Considering a vertical line (intersects each solvent line at the same elegant strength) at 48% methanol intersects the acetonitrile at 38%. Use acetonitrile/water instead of 48% methanol water 52%, the good starting percentage of acetonitrile must be 38%.

Step by step solution

01

Definition of isocratic elution

When the mobile phase has a constant concentration, isocratic elution is a term used in chromatography where the concentration of the mobile phase is constant throughout the chromatographic process.

02

Percentage of acetonitrile

Considering a vertical line (intersects each solvent line at the same elegant strength) at 48% methanol intersects the acetonitrile at 38%. Use acetonitrile/water instead of 48% methanol water 52%, the good starting percentage of acetonitrile must be 38%.

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Most popular questions from this chapter

Morphine and morphine 3-b-d-glucuronide were separated on two different 50 3 4.6 mm columns with 3-mm particles.61 Column A was C18-silica run at 1.4 mL/min and column B was bare silica run at 2.0 mL/min.

(a) Estimate the volume,Vm, and time,tm, at which unretained solute would emerge from each column. The observed times are 0.65 min for column A and 0.50 min for column B.

(b) Column A was eluted with 2 vol% acetonitrile in water containing 10 mM ammonium formate at pH 3. Morphine 3-尾-d-glucuro-nide emerged at 1.5 min and morphine at 2.8 min. Explain the order of elution.

(c) Find the retention factor k for each solute on column A, usingtm5 0.65 min.

(d) Column B was eluted with a 5.0-min gradient beginning at 90 vol% acetonitrile in water and ending at 50 vol% acetonitrile in water. Both solvents contained 10 mM ammonium formate, pH 3. Morphine emerged at 1.3 min and morphine 3-b-d-glucuronide emerged at 2.7 min. Explain the order of elution. Why does the gradient go from high to low acetonitrile volume fraction?

(e) From Equation 25-12 in Box 25-4, estimate k* on Column B assuming S = 4 and withtm5 0.50 min.

  1. Use equation 25-1to estimate the length of a column required to achieve1.0脳104plates if the stationary phase particles size is10.5,5.0,3.0,or1.5渭尘

  2. If the retention time was 20mins on the 10.0渭尘 particle size column, what is the retention time on the 5.0,3.0,or1.5渭尘columns from part (a)? Assume that flow rate is constant for all columns.

  3. Use equation25-2to estimate the pressure of the column in (a) given that the pressure of the10.0渭尘column was4.4Mpa

  4. If the flow rate was2.0mL/min , what is the baseline width for the peaks on 10.5,5.0,3.0,or1.5渭尘columns form part (a)?

  5. Which of these column configurations would require a UHPLC instrument?

(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.

What does it mean for a separation procedure to be 鈥漴ugged鈥� and why is it desirable?

A known mixture of compounds A and B gave the following HPLC results:

A solution was prepared by mixing 12.49mgof Bplus 10.00mLof unknown containing just and diluting to 25.00mL. Peak areas of 5.97and 6.38were observed for AandB, respectively. Find the concentration of A(mg/mL)in the unknown.
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