HPLC Analysis of Amino Acids and Peptides Fundamentals and More

1
HPLC Analysis of Amino Acids and
PeptidesFundamentals and More

• Presented by Dr. Steven A. Cohen
• Principal Scientist
• Waters Corporation

2
Topics

• Advances in amino acid analysis
• Introduction
• Pre- and post-column methods
• Case study a detailed look at AQC chemistry
• Modern peptide analysis
• Analytical to microbore separations
• Optimizing separations
• Column advances for peptide separations
• Introduction to capillary HPLC

3
Amino Acid Structures
4
Amino Acid AnalysisSources and Samples Types

• Purified peptides and proteins
• Generated by chemical or enzymatic hydrolysis
• Crude Hydrolysates
• Grains/agricultural products
• Infant formulas
• Tissue hydrolysates
• Nutritional research centers, QC, food/feed
  companies
• Intravenous Solutions
• Tissue Culture Media/Fermentation Broths
• Process development, manufacturing QC
• Biopharmaceutical companies, media suppliers
• Biological fluids
• Metabolites and building blocks
• Neurotransmitters

5
Importance of Amino Acid Analysis

• Structural protein building blocks
• Composition is the number of AA/molecule
• Used for protein quantitation
• Key metabolic intermediates
• Clinical importance
• Nutritional studies
• Nutritive value (due to above factors)
• Animal feed
• Intravenous solutions
• Infant formulae
• Pet food
• Plant physiology

6
Methods for Amino Acid Analysis

• Separation of free amino acids
• Usually requires post-column derivatization
• Ion-exchange using strong cation exchange columns
• Ion-exchange using strong anion exchange columns
  (electrochemical detectiowithout derivatization
• Ion-pairing reversed phase HPLC
• Separation of derivatized amino acids
• Numerous derivative choices
• All separations by RPLC

7
Ideal Method

• Accurate, reproducible results
• Linear response range
• Reactive with both primary and secondary amino
  acids
• Single, stable derivative for each amino acid
• Fast analysis
• Easy sample preparation
• Sensitive detection
• Rugged method
• Minimal matrix effects
• Suitable for many types of samples

8
Advantages of Free Amino Acid Separations
(Ion-exchange)

• 50 years of experience, methods development and
  applications
• Post-column derivatization need not be
  quantitative
• Derivatization can be less sensitive to matrix
  components

9
Detection for Post-Column AAA

• Ninhydrin
• Primary and secondary AA's
• Colored products 570 nm for primary AA's,
• 440 nm for secondary AA's
• Sensitivity in the 10's of picomoles
• OPA
• Primary AA's
• Secondary AA's only if samples are oxidized
  (hypochlorite) prior to derivatization
• Sensitivity to 10 pmol

10
Ninhydrin Chemistry
11
Some IEX Disadvantages

• Columns can be poisoned by sample components
• Slow analysis
• Less sensitive than pre-column derivatization
• Traditional methods use dedicated equipment
• Need fast reactions
• Reagent must be neutral to the detection method

12
Methods for Pre-Column Derivatization

• Ortho-Phthalaldehyde (OPA)
• Phenylisothiocyanate (PITC, Pico-Tag AAA)
• Dimethylamino-azobenzyl sulfonyl chloride
  (Dabsyl-Cl)
• Fluorenylmethyl-o-chloroformate (FMOC-Cl)
• OPA FMOC-Cl (AminoQuant)
• 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate
  (AQC or AccQ-Tag AAA)
• Lots more, but none used frequently

13
Common Post-column Reagents
14
OPA Chemistry
15
Derivatization Chemistry for AQC
16
Emission Spectra of AQC-Ala and AMQExcitation _at_
250 nm Solvent Acetonitrile
17
Optimizing the Separation of an Amino Acid
MixtureKey Parameters for Altering Selectivity

• Solvent considerations
• Acetonitrile, methanol
• Aqueous eleunt
• Buffer composition and concentration acetate,
  phosphate
• Buffer pH
• Ion-paring reagents TFA, TEA, hexane sulfonic
  acid
• Misc. additives EDTA, azide
• Gradient profile
• Column temperature

18
Chromatography of Hydrolyzate Standard
Eluent A 140 mM sodium acetate, pH 5.50 7 mM
TEA Plus azide (1 - 10 ppm) and 5 mM EDTA Eluent
B Acetonitrile Eluent C Water Column
temperature 37C Gradient Time Flow A B Init
ial 1.0 100 0 0.5 1.0 99 1 17 1.0 94 6 28 1.0 8
3 17 33 1.0 83 17
19
Evaluating the Method

• Separation quality
• Resolution
• Noise
• Chromatographic performance
• Retention reproducibility
• Ruggedness
• Quantitative parameters
• Reproducibility
• Linearity
• Sensitivity
• Accuracy

20
Chromatographic Reproducibility
21
Method ReproducibilityAmount analyzed 100pmol,
n 10
RSD
ASP
GLU
HIS
THR
PRO
TYR
MET
ILE
PHE
Average CV
SER
GLY
ARG
ALA
CYS
VAL
LYS
LEU
Amino Acids
22
Effect of Buffer Constituentson Derivatization
Efficiency
Yield
Amino Acids
23
Analysis of Bovine Serum Albumin
24
Evaluating Quantitative Accuracy in Amino Acid
Analysis

• Comparing the derived composition to a
  theoretical one
• Calculate composition using Best Fit method
• Minimizes average errorError 100 abs((exp
  - actual)/actual)Average error (? Error)/ N

25
Compositional Analysis of Bovine Serum Albumin
Error
Residues per Mole
ASP
GLU
HIS
THR
PRO
VAL
LYS
LEU
SER
GLY
ARG
ALA
TYR
MET
ILE
PHE
Amino Acids
26
Compositional Accuracy for BSA
27
Compositional Analysis of b - Lactoglobulin A
28
Comparison of Derivatizing Reagents
29
Cell Culture Separation Standard
30
Typical Cell culture AnalysisSpent Media
31
Separation for Collagen Analysis
32
Separation of Neurotransmitter AA Standards at
40C
33
Basal Amino Acid Levels in Rat Brain
34
Analysis of Peptides by HPLC

• What are peptides?
• Where do we get them?
• Why do we analyze them?
• Optimizing separation methods
• Detecting peptides

35
Peptides Are ....

• Linear polymers of amino acids
• Linked head-to-tail via peptide bond (a form of
  amide bond)
• Protein fragments
• Size may range from 2 - 50 or so AA's in length
  or MW 150 - ca. 5000

36
Peptide Sources

• Protein fragmentation
• Enzymatic digestion
• Partial hydrolysis
• Synthesis
• Isolation from natural sources
• Protein fragmentation in vivo

37
Why Are We Interested?

• Bioactivity
• Hormones and cellular regulation
• Used to characterize parent proteins
• Potential as drugs
• Analogs
• Peptide mimetics

38
Structure of Peptides in Solution Neutral pH
39
Structure of Peptides in Solution Ion
Suppression (acid pH)
40
Use of Polar (Acid) Modifiers in Peptide
Separations

• Aids in peptide solvation
• Suppresses ionization
• Provides ion pairing
• Preferably volatile for sample prep and LC/MS

41
Structure of Peptides in Solution Ion
Suppression with Ion Pairing
42
Structure of Peptides in Solution Ion
Suppression with HCl
43
Detecting Peptides

• General Detection
• Low UV (peptide bond absorbance), 200 - 230 nm
• LC/MS
• Derivatization
• Selective Detection
• Side chain chromophore Trp, Tyr, Phe
• Fluorescence Trp and occasionally Tyr

44
Requirements for Analytical and Micropreparative
Peptide HPLC

• Good detection sensitivity
• Good efficiency
• Good recovery
• Volatile mobile phase

45
Why use Trifluoroacetic Acid (TFA)?

• Good detection sensitivity
• Good efficiency
• Good recovery
• Volatile mobile phase

46
Why Not Use TFA?

• Detection/baselines
• Acetonitrile gradients
• Mixing as a function of system volume

47
Replicate Injections of Peptide
MixtureCytochrome Tryptic Digest
48
Retention Data (n 10) For Cyt C
49
Baseline Noise Reduction Using Variable Stoke
Volume
Flow rate 0.5 ml/min
50ul Stroke
0.008
25ul Stroke
AU 214nm
100ul Stroke
0.004
0.00
20
60
40
Minutes
50
Bovine Cytochrome c Tryptic Map
51
Alternate Ion Pairing ReagentsTFA vs. HCl
52
Dilute HCl as a Mobile Phase Modifier

• Alternative Selectivity to TFA
• Volatile to facilitate sample recovery
• Optical Clarity
• Less background absorbency
• Spectral information

53
High Sensitivity Peptide Mapping with 6 mM
HClTryptic Digest of Chicken Cytochrome c (8
pmol)
54
Alternate Ion Pairing ReagentsTFA and HFBA
Sample Rabbit Cytochrome c
tryptic digest 500 pmol
Column DeltaPak C18 5um,
300A

2 mm x 150 mm
Eluents Awater/ 0.1 TFA or
HFBA
Bacetonitrile/ 0.1 TFA
or HFBA
Gradient O-60 B 120 min
Flow 0.18 ml/min
Temp 35 C
55
Peptide Mapping with TFA200 nm vs. 214 nm
56
Peptide Mapping with HCl200 nm vs. 214 nm
57
Comparison of 214 vs 280nmDetection of Aromatic
Amino Acids
58
Two Wavelength DetectionSelective Heme Detection
59
Influence of TFA Concentration on Separation
Selectivity
0.05 TFA in solvents A and B
10
13
9
12
5 6
2
4
11
8
1
3
7
0.1 TFA in solvents A and B
13
10
9
12
5,6
2
4
8
11
1
7
3
10
0.2 TFA in solvents A and B
13
5,6
12
9
2
8
11
4
1
7
3
60
Optimizing TFA in Both Eluents
0.05 TFA in solvents A 0.1 TFA in solvent B
0.1 TFA in solvents A and B
61
Influence of Temperature on Selectivity
62
Selectivity Modification with Bonded
PhaseSymmetry C18 vs. SymmetryShield
Shield Chemistry Polar (carbamate) group near
surface Less hydrophobic packing
63
Why is Batch-to-Batch Reproducibility Important

• Validated RP-HPLC assays require HPLC columns to
  be reproducible from column-to-column and
  batch-to-batch.
• Columns which perform reproducibly in terms of
  selectivity and separation characteristics from
  batch-to-batch ensures reliable, reproducible and
  robust assays over the life of the product.

64
An Example of Batch-to-Batch Reproducibility
Batch 107
Batch 106
Batch 104
Batch 103
10
30
50
Minutes
65
Alternate Selectivity with a Organic-Silica
Hybrid Packing (XTerra)

• Not traditional silica
• Includes a hydrophobic component (CH3 groups)
  embedded in the (largely) silica particle
• Retains many of the favorable silica properties
• Improves key properties
• Higher pH stability
• Better temperature stability
• Better peak shape for basic components
• Compatible with peptide separations?

66
Symmetry vs. Xterra Columns
Column Dimensions 1 x 150 mm Flow Rate 50
ml/min C18, 100 A, 3.5 um 3 B to 43 B in 80
minutes A 0.05 TFA, B 0.043 TFA in MeCN
Symmetry
0.05
AU
0.00
0.05
XTerra
AU
0.00
10
20
30
40
50
60
70
Minutes