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Demystifying the Chromatographic Process

Agilent Technologies, Inc.Sept 12, 2012

Bill Champion800-227-9770, opt 3, opt 3, opt [email protected]

Sept 12, 2012

Demystifying Chromatographic Process Agilent Restricted

Objectives of Talk

• Chromatography is a physical process

• Much can be described with simple equations

• Understanding the process simplifies

• Understanding the process simplifies Method development, Troubleshooting, Predicting behavior, etc.

Sept 12, 2012


The first question may seem a bit silly, but some years ago we received a call from a customer (Big Pharma) seeking help with a method problem. They had tried everything to no avail. During our conversations we determined that the system was working properly, but a sample had failed. The first bad sample in five years of production. The first reaction was that it had to be the method. Wrong! That is why it is important to ask questions…the Right questions and get answers.Answering the questions here will lead you to the answer.

Topics

• Chromatographic Process

• What Affects Resolution, Rs• What Affects Retention, tR

• What Affects Pressure, P

• Mis-use, Over-interpretation of Chromatographic Equations

Sept 12, 2012



Conclusions of Talk

Effect of Increasing

N α k tR P

Flow Van Deemter No No Increase Increase

% B Slight Varies Exp Dec Exp Dec Varies

Temperature (T) Increase Varies Decrease Decrease Decrease

Particle Size (dp) Decrease No No No Decrease

Sept 12, 2012


Col Length (L) Increase No No Increase Increase

Col Diameter(d) Slight No No No* No*

Pressure (P) No No No No

* At constant linear velocity


Demystifying the Chromatographic Process

They said there WOULD be math!

Agilent Technologies, Inc.Sept 12, 2012

Bill Champion800-227-9770, opt 3, opt 3, opt [email protected]

Sept 12, 2012


Chromatographic Process

• Partition between mobile phase and stationary phase

• Description of the separation:

Sept 12, 2012




• Partition between mobile phase and stationary phase (K = Cs/Cm)

• Description of the separation:

Sept 12, 2012




• Partition between mobile phase and stationary phase (K = Cs/Cm)

• Description of the separation:Rs – Resolution

Rs – ResolutionN – Column Efficiency, Platesk, k’ – Retention Factor, Capacity Factor α – Selectivity

Sept 12, 2012



Topics

• Chromatographic Process

• What Affects Resolution, Rs

• What Affects Retention, tR


Sept 12, 2012



Definition of Resolution

Rs =∆tR

w

Resolution is a measure of the ability to separate two components

Sept 12, 2012


Resolution is a key term. “Resolution” is often the objective of using a separation technique such as HPLC. Resolution is a defined term. It is a measure of ability to distinguish between or to separate two components. More specifically, Resolution is defined as the difference in retention divided by the average of the peak widths. This equation shows that resolution increases if the two peaks have large differences in their retention times or if the peaks are narrow. In many of the following slides we will show how we try to increase the separation of the peaks or decrease the peak widths to improve resolution.

Definition of Resolution

Rs =tR-2 - tR-1

(w2 + w1)/2=

∆tR

w

Resolution is a measure of the ability to separate two components

Sept 12, 2012


Resolution is a key term. “Resolution” is often the objective of using a separation technique such as HPLC. Resolution is a defined term. It is a measure of ability to distinguish between or to separate two components. More specifically, Resolution is defined as the difference in retention divided by the average of the peak widths. This equation shows that resolution increases if the two peaks have large differences in their retention times or if the peaks are narrow. In many of the following slides we will show how we try to increase the separation of the peaks or decrease the peak widths to improve resolution.

Resolution … Determined by 3 Key Parameters –

Efficiency, Selectivity and Retention

The Fundamental Resolution Equation

w∆tR

=


Sept 12, 2012

N = Column Efficiency – Column length and particle size

α α α α = Selectivity – Mobile phase and stationary phase

k = Retention Factor – Mobile phase strength

w

With substitution and some simplifying assumptions, we can get an equation that shows the relationship of Resolution to some other fundamental chromatographic parameters. This is useful in understanding how to improve the separation. I will define and discuss these terms in the next few slides. Increasing N, sharpens the peaks - we can increase N by increasing column length or decreasing particle size Increasing k, increases the retention and typically increases the separation - we can increase k by decreasing mobile phase strength – which in reversed-phase chromatography, means decreasing the amount of MeOH or ACN in the mobile phase. Increasing a, increases the separation - we can increase a by adjusting mobile phase, that includes % organic including pH, temp, type of solvent or we can use a different stationary phase. Increasing N or k, are brute force approaches – increasing a is more subtle and often requires experimentation and understanding The next slide shows the definition of these terms and we will discuss them further.




w∆tR

=2


Sept 12, 2012




w

cf, Foley, Analyst, 116, 1275 (1991)





w∆tR

=1

11


Sept 12, 2012




w

cf, Foley, Analyst, 116, 1275 (1991)

11


k

Factors that Improve Resolution….

.w

∆tR=

N

a


Sept 12, 2012

k

α

N

Increaseretention

Change relativepeak position

Reduce peakwidth

This slide is a graphical presentation of resolution. And it shows how we might improve resolution. We can increase the retention (measured as k’), we can improve the difference in the separation (as measured by a)we can improve the column efficiency (as measured as N); we can improve the difference in the separation (as measured by a). We will define and discuss these terms in the next few slides.

Chromatographic ProfileEquations Describing Factors Controlling RS

k =(tR-t0)

t0

Selectivity

Retention Factor


α = k2/k1

N = 16(tR / tW)2

Theoretical Plates-Efficiency

Selectivity

Sept 12, 2012

The separated components are visualized using a detector (UV, RI or fluorescence) and we have a chromatographic profile with each component seen as a peak. So in this profile we have three components, A, B and C. Dead time = t0 = Vm/F This shows the terms in the resolution equation from the previous slide. K is a measure of retention based on the number of column volumes required to elute the peaks from the column. This is a fundamental parameter. Were there time, I would discuss this in much more detail. Selectivity is defined as the ratio of the retention factors. This means it is the ratio of the column volumes required to elute the two components N is the number of theoretical plates. There are several ways to calculate N for well behaved, Gaussian peaks. 16 (tr/w)^2 is the shown in the chromatogram for Peak B. It is usually easier to measure the peak at half-height, so we often use the second form, 5.54 (t/w05)^2. Notice that as the retention times increase that the peaks get broader – that also means they get shorter.

Some Basic Chromatography Parameters

• Resolution (Rs)

• Retention Factor (k), Capacity Factor (k’)

• Selectivity or Separation Factor (α)


• Column Efficiency as Theoretical Plates (N)

Sept 12, 2012


Retention Factor (k), Capacity Factor (k’)

Chromatographic separation is an Equilibrium Process

Sample partitions between Stationary Phase andMobile Phase:

K = Cs/Cm

Compound moves through the column only while inmobile phase.

Separation occurs in Column Volumes. (Flow is volume/time – mL/min)

Sept 12, 2012


Retention Factor is a fundamental measurement of the separation. The separation is an equilibrium process – the analyte partitions between mobile phase and stationary not unlike a component partitioning between water and hexane in a separatory funnel. A constant, the Partition Coefficient, is a measure of the ratio in the two phases. In the chromatographic process, as the amount of analytie in the mobile phase (Cm) increases, the amount in the stationary phase (Cs) increases proportionally. If we account for the volumes of the phases and some other issues, we get a parameter related to the Partition Coefficient, k, which calculated as the solvent volume that the analyte is retained in


k is measure of number of column volumes required to elute compound (proportion of time in stationary phase)..

k =t0

tR – t0K = Cs/Cm =>=>

Fundamental, dimensionless parameter that describesthe retention (independent of flow rate, column length).

k = 1 to 20 - OK; k = 3 to 10 - Better; k = 5 to 7 - Ideal

Sept 12, 2012



Measure of number of column volumes required to elute compound – fraction of time spent in SP (Cs)

k = =(VR – V0)

V0

(tR – t0)

t0

elute compound – fraction of time spent in SP (Cs)

Column Vol (V0)


Sept 12, 2012


Un-retained component – elutes w/ solvent front, Cs= 0

Cs = 0 => k = 0

Column Vol (V0)

1


Sept 12, 2012


Un-retained component – elutes w/ solvent frontk = (1 - 1) / 1 = 0

Column Vol (V0)

1


k = (tR – t0) / t0

Sept 12, 2012


Component retained – elutes in 1 add’l column volumesk = (2 - 1) / 1 = 1

Column Vol (V0)

21

k = (tR – t0) / t0


Sept 12, 2012



Column Vol (V0)

31 2

k = (tR – t0) / t0


Sept 12, 2012



Column Vol (V0)

841

k = (tR – t0) / t0

2 3 5 76


Sept 12, 2012



k = 1 to 20 - OK; k = 3 to 10 - Better; k = 5 to 7 - Ideal

Column Vol (V0)

841

k = (tR – t0) / t0

2 3 5 76


Sept 12, 2012

k


.w

∆tR=

(t -t )

N

a


Sept 12, 2012

k

α

N

Increaseretention


Reduce peakwidth

k =(tR-t0)

t0



Selectivity

Retention Factor

k =(tR-t0)

t0


α = k2/k1

N = 16(tR / tW)2


Selectivity

Sept 12, 2012



• Resolution (Rs)





Sept 12, 2012


Selectivity (α)

α is measure relative difference in retention

α =k1

k2

Sept 12, 2012


Selectivity (α)

α is measure of relative difference in retention

By definition, k is more retained component;

α =k1

k2

By definition, k2 is more retained component;k1 is less retained component, so α is always ≥ 1

To obtain separation, α must be > 1

Sept 12, 2012


k


.w

∆tR=

N

a


Sept 12, 2012

k

α

N

Increaseretention


Reduce peakwidth

α = k2/k1

Selectivity



k =(tR-t0)

t0

Retention Factor

Selectivity


N = 16(tR / tW)2


α = k2/k1

Selectivity

Sept 12, 2012


Selectivity

• Mobile Phase

• Stationary Phase

Sept 12, 2012



Different Mobile Phases May Give Different Selectivity

min0 2 4 6 8 10 12 14 16 18

mAU

0

100

200

300

400

500

600

700

50/50 MeOH/HOH

Toluene ...

... AnisolePhenethanol ...

p-F-Phenethanol ….....

mAU700

OMe


Sept 12, 2012

min0 2 4 6 8 10 12 14 16 18

0

100

200

300

400

500

600

700

41/59 ACN/HOH

ZORBAX® SB-C18 4.6 x 250 mm1 mL/min, 40°C, 225 nm

OH

Selectivity

• Mobile Phase

• Stationary Phase

Sept 12, 2012


Different Stationary Phases May Give Significantly Different Selectivity

min0 2 4 6 8 10 12

mAU

0

100

200

300

mAU

0

100

200

300

Columns: Zorbax StableBond, 3.0 X 100 mm, 3.5-um

Mobile Phase: 38% methanol in 0.4% formic acid

Zorbax SB-CN

Zorbax SB-AQ

1 2 3 45

6

7

1 2 3 4 7

6

5

O

OH

OH

OHOH

OH

(+)-Catechin

O

OH

OH

OHOH

OH

(-)-Epicatechin

OO

OOH

OH

O

O

OH

OH

OH

OOH

OH

OH

CH3

Naringin

O

O

OOH

OHOH

OH

O

OH

OH

OH

OOCH3

OH

OH

OH

Rutin


Sept 12, 2012

min*0 2 4 6 8 10 12

min*0 2 4 6 8 10 12

mAU

0

100

200

300

min*0 2 4 6 8 10 12

mAU

0

100

200

300

Zorbax SB-Phenyl

Zorbax SB-C18

1 2

34 7

6

5

1 2 3 4 7

65

O

O

OH

OH

OH

OH

OH

Quercetin

O

OOH

OH

OH

4',5,7-Trihydroxyflavanone

OH

CH3CH3

CH3

Thymol

Flavanones

This slide answers 2 questions people have. First, does having different bonded phases really make a difference. This separation show that having all these choices is important and will be important for many customers. All he C18’s were tried with the same mobile phase and I have arranged the chromatograms in one possible order for how they could be chosen. Eclipse Plus is definitely the first choice. Everything is resolved and the peak shape and efficiency are good. I have StableBond SB-C18 as the second choice here. It provides a very different selectivity because SB columns are not endcapped. This is a good reason to try a StableBond column. With this mobile phase of mostly acetonitrile this is an ok choice. It is a good strategy. I have Eclipse XDB-C18 as the 3rd choice. Resolution could likely be achieved on the Eclipse XDB column, but the results would not be any better than Eclipse Plus, so it is a 3rd choice. I have Extend-C18 as the 4th choice. The separation is not going to be achieved on this column and since it is not a high pH application, there is no reason for Extend to be selected for this application. These are arranged in the order that I would choose the columns and this is a general recommendation for many separations. For the RRHT columns you can see why it is a benefit to have choices for separations and that it is important for Agilent to have the new materials like Eclipse Plus and the older materials – SB, Eclipse XDB, ad Extend – available in 1.8um RRHT as well.

Similar Stationary Phases May Give Different Selectivity

Eclipse Plus C18

StableBond SB-C18

Mobile phase: (69:31) ACN: waterFlow 1.5 mL/min.Temp: 30 °CDetector: Single Quad ESI positive mode scan Columns: RRHT 4.6 x 50 mm 1.8 um

1 23

4

1 2 3 4 5

1 234

1st choiceBest Resolution & Peak Shape

2nd choiceGood alternate selectivity due to non-endcapped


Sept 12, 2012

Eclipse XDB-C18

Extend-C18

Sample:1. anandamide (AEA)2. Palmitoylethanolamide (PEA)3. 2-arachinoylglycerol (2-AG)4. Oleoylethanolamide (OEA)

1 23

4

1 2 3 4 5

1 2,3 4

1 2 3 4 5

��1 2 3 4 5

3rd choiceGood efficiency & peak shapeResolution could be achieved

4th choiceResolution not likely,Other choices better, for this separation.

Multiple bonded phases for most effective method development.Match to one you’re currently using.

This slide answers 2 questions people have. First, does having different bonded phases really make a difference. This separation show that having all these choices is important and will be important for many customers. All he C18’s were tried with the same mobile phase and I have arranged the chromatograms in one possible order for how they could be chosen. Eclipse Plus is definitely the first choice. Everything is resolved and the peak shape and efficiency are good. I have StableBond SB-C18 as the second choice here. It provides a very different selectivity because SB columns are not endcapped. This is a good reason to try a StableBond column. With this mobile phase of mostly acetonitrile this is an ok choice. It is a good strategy. I have Eclipse XDB-C18 as the 3rd choice. Resolution could likely be achieved on the Eclipse XDB column, but the results would not be any better than Eclipse Plus, so it is a 3rd choice. I have Extend-C18 as the 4th choice. The separation is not going to be achieved on this column and since it is not a high pH application, there is no reason for Extend to be selected for this application. These are arranged in the order that I would choose the columns and this is a general recommendation for many separations. For the RRHT columns you can see why it is a benefit to have choices for separations and that it is important for Agilent to have the new materials like Eclipse Plus and the older materials – SB, Eclipse XDB, ad Extend – available in 1.8um RRHT as well.

Selectivity

• “Chromatography is an Experimental Science”

• α not as predictable as k, N – but more powerful

Sept 12, 2012



• Resolution (Rs)





Sept 12, 2012


Column Efficiency (N)

N - Number of theoretical plates.

“Plates” is a term inherited from distillation theory. It is a measure of the relative peak broadening (or peak width) for an analyte in a separation – w

N = 16tR 2


Sept 12, 2012

N = 16tRw

A Number of Theoretical Plates



We can increase N by increasing the length of the column or decreasing the size of the stationary phase particles.(1.8 µm > 2.7 µm > 3.5 µm > 5 µm > 10 µm)

N = 16tR 2

= f(L, 1/d )


Sept 12, 2012

N = 16tRw

L =dp =

= f(L, 1/dp)

column lengthparticle size



We can increase N by increasing the length of the column or decreasing the size of the stationary phase particles.(1.8 µm > 2.7 µm > 3.5 µm > 5 µm > 10 µm)


Sept 12, 2012

L =dp =

column lengthparticle size

p

Test Chromatogram


Sept 12, 2012 Slide 46

Van Deemter Curve Factors Affecting N

Pla

te H

eig

ht

H (

L/N

)

H = A + B/u + C u

Large Particle

SmallParticle

H = L/N


Sept 12, 2012

Pla

te H

eig

ht

H (

L/N

)

Linear Velocity u

The smaller the plate height, the higher the plate number and the greater the chromatographic resolution

Resistance to Mass Transfer

Putting it Together The van Deemter Equation

Pla

te H

eig

ht

H

Sum Curve: van-Deemter H = A + B/u + C u

Large Particle

Small

Particle

H = L/N


Sept 12, 2012

Longitudinal diffusion

Pla

te H

eig

ht

H

Linear Velocity u

Eddy Diffusion

Resistance to Mass Transfer

The smaller the plate height, the higher the plate number and the greater the chromatographic resolution

u opt

H min

This slide shows the van Deemter plot and is importance to chromatography. You can get better diffusion through smaller particles. Here [indicated H equation],we have the equation for the height equivalent of theoretical plates. It is is defined as column length[indicate L] divided by the number of theoretical plates[indicate N]. This comes from distillation theory, and allows us to separate parameters easily. We will discuss this in greater detail in a moment. We want this number to be as small as possible.

Van Deemter Curve Effect of Particle Size

0.0015

0.0020

0.0025

0.0030Column: ZORBAX Eclipse XDB-C18 Dimensions: 4.6 x 50 mmEluent: 85:15 ACN:WaterFlow Rates: 0.05 – 5.0 mL/minTemp: 20°CSample: 1.0µµµµL Octanophenone in Eluent

HE

TP

(cm

)

5.0µµµµm3.5µµµµm

H = A + B/u + Cu

5.0 µµµµm

3.5 µµµµm


Sept 12, 2012

0.0000

0.0005

0.0010

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

Volumetric Flow Rate (mL/min)

HE

TP

(cm

)

3.5µµµµm1.8µµµµm

Smaller particle sizes yield flatter curves, minima shift to higher flow rates

3.5 µµµµm

1.8 µµµµm

So first let’s look at a Van Deemter curve. On the y-axis we measure HETP – height equivalent of a theoretical plate – basically the smaller the better – the more plates in the column. What it is also telling you is how broad the peaks are. Because broad peaks have low efficiency, we want the narrowest peaks possible. So we need to design columns that make the sharpest peaks. On the x-axis we have linear velocity (u) usually, but often substituted with volumetric flow rate because it is easy to plot. The typical relationship when plotted is a curved relationship with a minimum and an almost linear increase in HETP with linear velocity. This means the peaks get broader when you use higher flow rates and we don’t want that. What is controlling this is the “C” term in the equation above – which talks about mass transfer. This is the movement of the analytes in and out of the pores in the particles while the mobile phase is flowing. Therefore if you have a smaller particle you should reduce this resistance to mass transfer and the slope of the line should become flatter. That is exactly what happens. We care about this because it means you can increase the flow rate and maintain efficiency, you would increase the flow rate to have a faster analysis. So you gain speed, but keep sharp peaks – that is the goal and it is something that a 1.8um particle accomplishes better than any other size currently available.

Columns Packed with Smaller Particles Provide Higher Efficiency

3 micron

N

sub 2 micron

N 1/(d )α

Large Particle

SmallParticle

5 micron

10 micron

Velocity

N

P 1/(dp)2α

N 1/(dp)α

Sept 12, 2012


Decreasing Particle Size

5 µm, 150 mm; 8 min, 170 bar

3.5 µm, 100 mm; 6 min, 204 bar

1.8 µm, 50 mm; 3.4 min, 300 bar

Sept 12, 2012


“Reversed-Phase HPLC Separation ofWater-Soluble Vitamins on AgilentZORBAX Eclipse Plus Columns”,5989-9313EN (2008)


k =(tR-t0)

t0

Selectivity

Retention Factor


α = k2/k1

N = 16(tR / tW)2

Theoretical Plates - Efficiency

Selectivity

Sept 12, 2012


k =(tR-t0)

t0

Selectivity

Retention Factor

tR


α = k2/k1

N = 16(tR / tW)2

N = 5.54(tR / tW0.5)2

Theoretical Plates - Efficiency

Selectivity

tW0.5

Sept 12, 2012


• Resolution (Rs)





Sept 12, 2012


k

Factors that Affect Resolution….

.w

∆tR=

N

a


Sept 12, 2012

k

α

N

Increaseretention


Reduce peakwidth


Resolution as a Function of Selectivity, Column Efficiency, or Retention

4.00

5.00

6.00

7.00

Re

sol

utio

n Increase N

Increase Alpha

Selectivity Affects Resolution Most• Change bonded phase

• Change mobile phaseα


Sept 12, 2012

0.00

1.00

2.00

3.00

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Re

sol

utio

n

Increase Alpha

Increase k'

Rs = N½/4 •••• (αααα-1)/αααα •••• k’/(k’+1)

Nk

Basically this slide takes the fundamental resolution equation – shown in large form on the previous slide – and plots the impact of the different parameters. This makes it easy for those who can’t visualize things from the equation itself. This plot shows that alpha or selectivity – is the most critical parameter.

Topics



Sept 12, 2012



Retention

• Mobile Phase Strength

• Temperature

Sept 12, 2012



Effect of Mobile Strength on Retention

k = 0.5

k = 0.8

k = 1.3

k =(tR-t0)t0

k = 1.3

k = 2.0

k = 3.4

Sept 12, 2012


Mobile Strength

k =(tR-t0)t0

Sept 12, 2012


Mobile Strength

Effect of %B on k

Sept 12, 2012


0

0.5

1

1.5

2

2.5

3

3.5

4

35 40 45 50 55 60 65

k

% ACN

k1

k2

Mobile Strength

Effect of %B on k

Sept 12, 2012


0

0.5

1

1.5

2

2.5

3

3.5

4

35 40 45 50 55 60 65

k

% ACN

k1

k2

k = (kw) e –S(%B)

Mobile Strength

Effect of %B on ln(k)

Sept 12, 2012


-1.50

-1.00

-0.50

0.00

0.50

1.00

1.50

2.00

2.50

35 40 45 50 55 60 65

ln(k

)

% ACN

ln(k1)

ln(k2)

Mobile Strength


Sept 12, 2012


y1 = -0.0996x + 4.88R² = 0.9999

y2 = -0.0942x + 4.976R² = 0.9994

-1.50

-1.00

-0.50

0.00

0.50

1.00

1.50

2.00

2.50

35 40 45 50 55 60 65

ln(k

)

% ACN

ln(k1)

ln(k2)

Mobile Strength


Sept 12, 2012


y1 = -0.0996x + 4.88R² = 0.9999

y2 = -0.0942x + 4.976R² = 0.9994

-1.50

-1.00

-0.50

0.00

0.50

1.00

1.50

2.00

2.50

35 40 45 50 55 60 65

ln(k

)

% ACN

ln(k1)

ln(k2)

a

Change in Order of Elution

-- k = 7.4

-- k = 20

-- k = 55ln(k) = ln(kw) – S(%B)

Sept 12, 2012


ACN0 30%7.5%/min

ACN0 20%8.65%/min

ACN0 20%2 min

10.65%/min

Sept 12, 2012


Using Higher Temperatures• Reduces Analysis Time• May Change Selectivity

Effect of Temperature

Sept 12, 2012


1/T (°K)


Topics



Sept 12, 2012



What About Pressure? Pressure Increases with Decreasing Particle Size

∆P = Pressure Drop

= Fluid Viscosityη

∆P =η L vθ dp

2 ü Many parameters influence column pressureüParticle size and column length are most criticalüLong length and smaller

Equation For Pressure Drop Across an HPLC Column


Sept 12, 2012

L = Column Length

v = Flow Velocity

= Fluid Viscosityη

= Dimensionless Structural Constant of Order 600 For Packed Beds in LC

θ

d = Particle Diameterp

üLong length and smaller particle size mean more resolution and pressureü We can now handle the pressure

Comparison of Effect of Water/ACN and Water/MeOH on Viscosity

1.001.201.401.601.80

cP

Viscosity (cP) - 25°C


Sept 12, 2012

0.000.200.400.600.80

0 10 20 30 40 50 60 70 80 90 100

cP

%B

MeOH 25°CACN 25°C

Topics



Sept 12, 2012



Conclusions of Talk

Effect of Increasing

N α k tR P

Flow Van Deemter No No Increase Increase

% B Slight Varies Exp Dec Exp Dec Varies

Temperature (T) Increase Varies Decrease Decrease Decrease

Particle Size (dp) Decrease No No No Decrease

Sept 12, 2012


Col Length (L) Increase No No Increase Increase

Col Diameter(d) Slight No No No* No*

Pressure (P) No No No No

* At constant linear velocity


Thank you – Questions?


Sept 12, 2012

Bill Champion800-227-9770, opt 3, opt 3, LC Column [email protected]

Column Connectors Used in HPLC

0.090 in.

0.130 in.

Swagelok Waters

0.090 in.

0.170 in.

Parker Rheodyne

Valco Uptight0.090 in.

0.080 in.

Troubleshooting LC Fittings, Part II. J. W. Dolan and P. Upchurch, LC/GC Magazine 6:788 (1988)

Sept 12, 2012


To continue with the concept of matched fittings, this slide shows various column connectors used in HPLC. Notice that for each vendor, the distance from the end of the tubing to where the ferrules are "seated" or "swaged" is different? If the distance is too great for the end fitting of the column, the ferrule will not seal with the column and the fitting will leak. If you then tighten down on the fitting, the tubing will puncture the column frit. If the distance is too short, you can seal the ferrule and not have a leak, but there will be a large extra column volume for band broadening to occur. Notice how the ferrules are shaped differently as well? Some are designed to be used with columns and some are specific to injectors or detectors or pumps. DO NOT INTERCHANGE THESE! They may become a permanent fixture, or imbedded in your column or equipment. The last point is, once you seat the ferrule or swage it into place, that fitting is the right size for only that column type. Each column end fitting or holder from one vendor is not machined the same as another vendor. Thread number and depth vary. A cartridge system with the same end holders for all cartridges prevents you from needing to have multiple dedicated end fittings for multiple columns. Many users are changing to universal end fittings which are plastic or PEEK, yet seal at high pressures, are adjustable, and re-usable. There is little chance of mismatch.

What Happens If Connections Are Poorly Made?

Ferrule cannot seat properly

Wrong … too long

Wrong … too short

If Dimension X is too long, leaks will occur

Mixing Chamber

If Dimension X is too short, a dead-volume, or mixing chamber, will occur

X

X

Sept 12, 2012


With either fitting type you need to make sure the connection is made correctly and that it does not slip! Go to next slide.

∆Φ ∆Φ ∆Φ ∆Φ = change in volume fraction of B solventS = constant

tg F

S ∆Φ∆Φ∆Φ∆Φ Vm

k* =

Gradient Retention (k*)Selectivity in gradient elution is determined by the gradient retention factor

k =t0

tR – t0


Sept 12, 2012

S = constantF = flow rate (mL/min.)tg = gradient time (min.)

Vm = column void volume (mL)

• S ≈≈≈≈ 4–5 for small molecules

• 10 < S < 1000 for peptides and proteins

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In gradient separation the effective value of k (k*) for different bands will be about the same.

1. Gradient Steepness Affects Retention (k*) and Resolution The last variable we’ll discuss today is gradient steepness. On this slide is shown the fundamental equation that relates gradient retention, k*, with gradient time, flow rate, gradient range, and column volume. You can think of gradient steepness, b, as being analogous to % organic modifier in isocratic separations. When gradient steepness is decreased, retention increases; when gradient steepness is increased, retention decreases. As gradient steepness is decreased, peak width increases and peak height decreases. Key things to remember: 3 As long as b is kept constant, the selectivity and resolution will remain the same in gradient separations. 3 Because different analytes have characteristic S values (slopes of log k vs. % organic and log k* vs. gradient time), decreasing gradient steepness does not necessarily mean better retention and resolution. 3 This is analogous to the statement that changes in % organic modifier do not produce the same changes in retention among different analytes. 3 For most small molecules (<500-1000 Da), S values are about 4-5. However, S values for peptides and proteins are much larger and vary from each other over 2-3 orders of magnitude. 3 Gradient separations are necessary for separating peptides and proteins, and small changes in gradient steepness cause large changes in retention. k’ (or k*, in a gradient) is a simple way to increase resolution. However; this is at the expense of longer runtime. In addition, peak width increases and peak height is reduced (making quantitation more difficult).

This Relationship Says that to Keep Relative Peak Position in the Chromatogram Unchanged

Column length Decrease in tG or F

Increase in ∆Φ

Any Decrease in Can be Offset by a Proportional


Sept 12, 2012

Column volume (i.d.)

∆Φ (same column)

Decrease in tG or F2

Increase in ∆Φ

Decrease in tG or F

k* =tG • F

S • ∆Φ • Vm

Sept 12, 2012


demystifying the chromatographic process - agilent...demystifying the chromatographic process...

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