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(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 150.46-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 151.0cmcolumn, 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.

Short Answer

Expert verified

The value of gradient timetG=28.6min

Value ofk* is same

k*=12.9;F=4.7mL/min,tG=11.5min

Step by step solution

01

Define gradient time

tG the gradient time (the time from the start to the end of the gradient), VM the column hold-up volume and F the mobile phase flow rate. The gradient steepness parameter will vary with the S-value, which is only roughly constant for similar compounds.

02

Step 2: Calculating Vm

We assume thatk* is 5 and S is 4 .The volume at which solvent front appear is:

Vm=L.dc22Vm=15cm.0.46cm22Vm=1.587mLrr

03

Step 3: Calculating tG  

We can calculatetGusing the following formula:

tGis

role="math" localid="1665031412999" k*=tG.F.Vm.StG=k*..Vm.SFtG=5.0.9.1.587mL.41mL/mintG=28.6min

04

Define gradient time and volume flow rate

tG the gradient time (the time from the start to the end of the gradient), VM the column hold-up volume and F the mobile phase flow rate. The gradient steepness parameter will vary with the S-value, which is only roughly constant for similar compounds.

Volume flow rate is the same as "Flux" and "R". There are two different equations used to express volume flow rate, it can be expressed as Volume/Time.

05

Calculating k* 

k* for optimized separation is:

k*=tG.F.Vm.Sk*=11.5min.1mL/min0.14.1.587mL.4k*=12.9

06

Checking the sample load 

If column is scale up to a151.0cmthe column has the same length as the previous column, but the diameter is increased from 0.46 to 1.0cm So, the volume increases by a factor of:

10.462=4.7

The flow of rate also is increased by the same factor:

The gradient time is unchanged at 11.5min

The sample loading is also increased by factor 4.7:

4.7.1mg = 4.7 mg

07

Verification of k*

Verification that k* is unchanged:

Vm=L.dc22Vm=15cm.1cm22Vm=7.5mLk*=tG.F.Vm.Sk*=11.5min.4.7mL/min0.14.7.5mL.4k*=12.9

k* is the same as in the previous column.

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Most popular questions from this chapter

what are criteria for an adequate isocratic chromatographic separation?

  1. Use equation 25-1to estimate the length of a column required to achieve1.0104plates 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?

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)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 = currentvoltage).

(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.

Microporous silica particles with a density of2.2g/mLand a diameter10渭尘of have a measured surface area of calculated the surface area of the300m2spherical silica as if it were simply solid particles what does this calculation tell you about the shape or porosity of the particles.

The 鈥渞ule of three鈥 states that the retention factor for a given solute decreases approximately threefold when the organic phase increases by10%.In figure 25-12,tm=2.7min. Find k for peak 5at 40%B .predict the retention time for peak at B and compare the observed and predicted times.

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